Hybridization and mismatch discrimination using oligonucleotides conjugated to minor groove binders

ABSTRACT

Conjugates between a minor groove binding molecule, such as the trimer of 1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate (CDPI 3 ), and an oligonucleotide form unusually stable hybrids with complementary target sequences, in which the tethered CDPI 3  group resides in the minor groove of the duplex. These conjugates can be used as probes and primers. Due to their unusually high binding affinity, conjugates as short as 8-mers can be used as amplification primers with high specificity and efficiency. MGB conjugation also increases the discriminatory power of short oligonucleotides, providing enhanced detection of nucleotide sequence mismatches by short oligonucleotides. The MGB-conjugated probes and primers described herein facilitate various analytic and diagnostic procedures, such as amplification reactions, PCR, detection of single-nucleotide polymorphisms, gene hunting, differential display, fluorescence energy transfer, hydrolyzable probe assays and others; by allowing the use of shorter oligonucleotides, which have higher specificity and better discriminatory power.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 08/415,370 (filed Apr. 3, 1995), the disclosure of which ishereby incorporated herein by reference in its entirety.

TECHNICAL FIELD

The present invention is in the field of molecular biology. Morespecifically, the invention is in the field of assays that utilizeoligonucleotides as primers or hybridization probes.

BACKGROUND

Minor groove binding agents which non-covalently bind into the minorgroove of double stranded DNA are known in the art. Intercalating agentswhich bind to double stranded DNA or RNA are also well known in the art.Intercalating agents are, generally speaking, flat aromatic moleculeswhich non-covalently bind to double stranded DNA or RNA by positioning(intercalating) themselves between interfacing purine and pyrimidinebases of the two strands of double stranded DNA or RNA. U.S. Pat. No.4,835,263 describes oligonucleotides which are covalently bound to anintercalating group. Such oligonucleotides carrying an intercalatinggroup can be useful as hybridization probes.

In many analytic, diagnostic and experimental systems in modern biology,oligonucleotides are used in procedures that require that they base pair(i.e., hybridize) with a nucleic acid sequence that is complementary tothe oligonucleotide. This hybridization process may be used to directlydetect a sequence in a nucleic acid molecule (i.e., probing), toinitiate synthesis at a specific sequence (i.e., priming), or to blocksynthesis by inhibiting primer extension (i.e., clamping). In all theseprocedures, the technique relies on the formation of a nucleic acidduplex (or hybrid) based on the principle that the duplex will form onlyif the two strands are complementary over a significant portion of theirlengths. Complementarity is determined by the formation of specifichydrogen bonds between the nucleotide bases of the two strands such thatonly the base pairs adenine-thymine, adenine-uracil and guanine-cytosineform hydrogen bonds, giving sequence specificity to the double strandedduplex. In a duplex formed between an oligonucleotide and anothernucleic acid molecule, the stability of the duplex is a function of itslength, the number of specific (i.e., A-T, A-U and G-C) hydrogen bondedbase pairs, and the base composition (ratio of guanine-cytosine toadenine-thymine or adenine-uracil base pairs), since guanine-cytosinepairs provide a greater contribution to the stability of the duplex thando adenine-thymine or adenine-uracil pairs.

Usually, the relative stability of a duplex is measured experimentallyby heating the duplex in solution until the strands of the duplexseparate. The quantitative stability of a duplex is expressed by thetemperature at which one-half the base pairs have dissociated, commonlyknown as the “melting temperature” or T_(m). In practice, this isusually measured by monitoring the ultraviolet absorbance of a solutionof nucleic acid while the temperature is increased and denoting theT_(m) as the temperature at half the maximal absorbance at 260 nm (sincean increase in absorbance at 260 nm accompanies the dissociation of thetwo strands of a duplex).

Essentially all procedures involving analysis of a target nucleic acidsequence require a hybridization step, either to determine directly ifthe complement of a known sequence (the probe) is present in a sample orto initiate synthesis (prime) from a specific sequence. Control of thespecificity of the hybridization step is key to successful and accuratenucleic acid analysis. In most cases, exact matching between thesequence of the probe or primer and the sequence of its target isrequired. Nevertheless, in some cases, the analytical approach requiresthe stabilization of a probe or primer in a duplex that is not a perfectmatch. Therefore, techniques and material that can be used to controlhybridization procedures such that it is possible, on the one hand, toobtain only perfectly matched duplexes and, under alternate conditions,to stabilize mismatched duplexes, would extend the use ofoligonucleotides and allow analytical and experimental procedures thatare now very difficult or unreliable.

For example, many analytical procedures require primer extension as ameans of amplifying or labeling a DNA or RNA sequence so that it may beexamined further. See, for example, Sambrook et al., MOLECULAR CLONING:A LABORATORY MANUAL, Second Edition, Cold Spring Harbor Laboratory Press(1989). These procedures include, but are not limited to,chain-termination sequencing-based on the Sanger Method (Sanger et al.(1977) Proc. Natl. Acad. Sci. USA 74:5463-5467), polymerase chainreaction (PCR) amplification of DNA or RNA sequences (U.S. Pat. Nos.4,683,202; 4,683,195 and 4,800,159; Mullis and Faloona; Meth. Enzymol.,vol 155, Academic Press, New York, 1987, pp. 335-50; and Saiki et al.(1985) Science 230:1350-1354), cDNA synthesis (Rougeon et al. (1975)Nucleic Acids Res. 2:2365-2378) and combinations of these procedures forspecific purposes such as “differential display” (Liang et al. (1992)Science 257:967-971), mRNA indexing (Kato et al. (1996) Nucleic AcidsRes. 24:294) and gene hunting (Tung et al. (1989) In Erlich, H. A.(ed.), PCR Technology: Principles and Applications for DNAAmplification. Stockton press, pp. 99-104) among others.

Each of these procedures requires hybridization, to a target sequence,of an oligonucleotide primer from whose 3′ terminus synthesis isinitiated. The ability of an oligonucleotide to serve as a primerdepends upon die stability of the duplex it forms with its template,especially at its 3′ terminus. The ability of an oligonucleotide toserve as a unique, specific primer depends upon the stability of theduplex its forms with its perfect complement and, conversely, on thelack of stability of a duplex including one or more noncomplementary(i.e., mismatched) base pairs. Current priming methods rely on the useof oligonucleotides sufficiently long to form stable duplexes attemperatures necessary or convenient for extension. However, longeroligonucleotides are more prone to mismatch pairing than shorteroligonucleotides. Further, specific information may restrict the use oflonger oligonucleotides.

To give one example, many methods involving oligonucleotides utilizesome type of amplification technology, often based on a polymerase chainreaction (PCR). See, for example, U.S. Pat. Nos. 4,683,195; 4,683,202;and 4,800,159. PCR has become an exceptionally powerful tool inmolecular biology, but certain factors limit its versatility. BecausePCR involves multiple cycles of DNA denaturation, elevated temperaturesare usually required, making the use of a thermophilic polymerizingenzyme necessary to avoid the inconvenience of supplying freshpolymerizing enzyme at each cycle. However, at the elevated temperaturesoptimal for activity of a thermophilic polymerase and required fordenaturation, oligonucleotides shorter than about 20 nucleotides(20-mers) do not form hybrids that are stable enough to serve as primersfor polymerase-catalyzed elongation. Consequently, current PCR-basedtechniques generally require primers at least 20 nucleotides in lengthto form hybrids that will be stable at the temperatures and stringenciescommonly used for PCR. Saiki (1989) In Erlich, H. A. (ed.), PCRTechnology: Principles and Applications for DNA Amplification. StocktonPress, pp. 7-16.

In another example, mRNA “indexing” requires priming from the 3′ end ofa messenger RNA (mRNA) molecule or from a cDNA made from the mRNA. Katoet al., supra. This technique employs separate populations ofoligo-dT-containing primers, each additionally containing an extensionof one to approximately three nucleotides adjacent to the oligo Asequence on the 5′ side of the oligo A. The objective is to causesynthesis of specific segments of DNA corresponding to the 3′ end ofeach mRNA (determined by the oligo A sequence) but separated intospecific populations, determined by the specific base at positions 1 to(approximately) 3, upstream of the oligo A. If each primer is used in aseparate reaction, separate populations of cDNAs are generated, each ofwhich is a subset of the total mRNA. These can be used to analyzecellular expression. This procedure is usually combined with PCR, byincluding a second primer in each separate reaction. The practicabilityof this method is limited by the necessity to use sufficiently longoligo thymidylate, complementary to the oligo A, to stabilize the firstprimer. This can result in stabilization of mismatches within the one tothree specific bases at the 3′ end of the primer. In a population ofprimer and templates, these mismatches allow synthesis of improperlyprimed, and therefore misleading cDNA molecules leading to incorrectindexing of mRNA. Alternatively, some primers are insufficiently stableto prime efficient synthesis; consequently, the extension products theywould have generated are underrepresented in the population, againleading to incorrect indexing of the corresponding mRNA. Short primersthat are stable at elevated temperatures commonly used for PCR, but thatform only perfect duplexes (i.e., do not prime mismatches) wouldincrease the utility of this technique.

Important new techniques, such as gene hunting and differential display,would also benefit from the use of shorter primers. In some cases, shortprimers are essential for these methods. In gene hunting, a family ofamplified transcripts shares a short degenerate sequence that specifiesa conserved peptide motif, and this priming sequence is necessarilylimited in length. Tung et al., supra. Stockton press, pp. 99-104. Indifferential display, complete representation of a transcript pool issought, and this is optimally achieved by priming with 6-mers. Theimpracticality of using such short primers necessitates the use oflonger degenerate ODNs. Liang et al., supra. However, long degenerateODNs may not provide an accurate representation of the complexity of amRNA population, since mispriming can generate non-specific products,and inefficient hybridization of the primer can lead to underrepresentation of certain transcripts. Buchner et al. (1995) Stat. Mol.Biol. 8:12-14. Application of longer oligonucleotides to viraldiagnostics are limited, because amplification of a common sequence frommultiple strains can be complicated by the presence of genomicvariability. Smits et al. (1992) J. Gen. Virol. 73:3263-3268. Again,shorter primers are desirable, since the shorter the sequence used forpriming, the less likely that it will encompass a region characterizedby genomic variability.

In addition to priming, oligonucleotide hybridization is used in severaltechniques to probe nucleic acid sequences. In general, these assaysrequire that the probes form perfectly-matched duplexes with targetsequences. These assays are usually based on one of three schemes: 1)The probe or target is labeled (e.g., with a radioactive isotope, afluorescent dye or a reactive compound), the nucleic acids are placedunder hybridization conditions following hybridization, thenon-hybridized labeled material is removed and the remaining label isquantitated. 2) The probe is specifically labeled and placed with thetarget DNA under hybridization conditions, following hybridization, thehybridized probe is detected by virtue of a property unique to a duplexcontaining the probe such as susceptibility to a duplex-specificnuclease (e.g., U.S. Pat. No. 5,210,015), 3) fluorescence generated byinteraction of a dye with duplex DNA (Wittwer et al. (1997)BioTechniques 22:130-138) or separating a fluorophore from a quenchingdye by the extension of the probe as a result of hybridization.

A method that could be used in essentially all these types of nucleicacid hybrid detection systems to enhance the distinction between exactduplexes and duplexes with one or more mismatched base pairs would be avery useful tool in specific nucleic acid sequence determination andclearly be valuable in clinical diagnosis, genetic research and forensiclaboratory analysis.

For example, many diseases are associated with known inheritedpolymorphisms or mutations. Many of these are due to single nucleotidechanges and, to be useful, a genetic assay based on hybrid formationmust be able to distinguish between a hybrid with all base pairs matchedand one with a single mismatch. A group of single base differences atcertain points in the sequences of human DNA called single nucleotidepolymorphisms have been determined to be stably inherited geneticmarkers (Schaeffer et al. (1998) Nature Biotechnology 16:33). Thesemarkers can be associated with ancestral populations and in some casescan be associated with characteristics such as disease susceptibility orresponse to environmental factors such as chemicals, drugs, etc.Although these polymorphisms can theoretically be discovered by thetedious process of gene sequencing, their use as genetic markersassociated with a phenotype in, for example, medical practice orresearch, necessitates a screening or typing system that is capable ofanalyzing DNA from tens to hundreds of individuals. This process willnot easily be accommodated by current methods of DNA sequencing. Singlenucleotide polymorphism analysis thus represents an additional field inwhich there exists a need for a reliable method for distinction ofsingle base differences in DNA sequences by a process such ashybridization.

Various additional assays that involve oligonucleotide priming are knownin the art. These include, but are not limited to, assays that utilizethe nuclease activity of a polymerase enzyme to release label from aprobe hybridized to an extension product (see, for example; U.S. Pat.No. 5,210,015), and assays in which hybridization of two or moreoligonucleotides to adjacent sites on a target nucleic acid results ininteractions between the oligonucleotides, such as, for example,fluorescence resonance energy transfer. See, for example,Stavrianopoulos et al., U.S. Pat. No. 4,868,103; and Heller et al.,European Patent Publication 070,685. These techniques are also limitedby the length of the oligonucleotide that can be used for efficienthybridization and/or priming. The ability to use shorteroligonucleotides would therefore be beneficial in these procedures and,indeed, in any application that involves hybridization of anoligonucleotide to a target nucleic acid.

Chemical modification of short oligonucleotides has been attempted, withan eye toward improving hybrid stability while retaining effectivepriming ability. Certain modifications, such as N3′→P5′ phosphoramidates(Gryaznov et al. (1994) J. Am. Chem. Soc. 116:3143-3144) and peptide(Nielsen et al. (1994) Bioconjugate Chem. 5:3-7) or guanidine (Dempcy etal. (1995) Proc. Natl. Acad. Sci. USA, 92:6097-6101) linkages, have beenshown to enhance hybrid stability. However, such modifiedoligonucleotides are non-extendible, because they lack a 3′-OH group,and are therefore unable to serve as primers. Other hybrid-stabilizingmodifications that have not been investigated with respect to theirability to support primer extension are 2′-modified sugars (Monia et al.(1993) J Biol. Chem. 268:14514-14522; Sproat et al. (1993) In Crooke, S.T. and Lebleu, B. (eds), Antisense Research and Applications. CRC Press,Boca Raton, Fla., pp. 352-362), conjugated intercalating agents(Asseline et al. (1984) Proc. Natl. Acad. Sci. USA 81:3297-3301) andsubstituted bases such as 2-aminoadenine (Lamm et al. (1991) NucleicAcids Res. 19:3193-3198) or C5 propynyl pyrimidines (Wagrier et al.(1993) Science 260:1510-1513). Thus, the need remains for a method ofmodifying short oligonucleotides so that they form more stable hybrids,such that the modification will not interfere with the ability of theoligonucleotides to serve as primers.

A further shortcoming in the use of oligonucleotides as probes andprimers is the difficulty of obtaining specificity such as singlenucleotide mismatch discrimination using oligonucleotide probes and/orprimers. In many cases, it is necessary to distinguish target sequenceswhich differ by a single nucleotide and, in some cases, it would bedesirable to do so using oligonucleotides. That is, it would be usefulto have a given oligonucleotide which is able to hybridize to a targetsequence with which it is complementary along its entire length (aperfect hybrid or perfect match), but which, under identical stringencyconditions, will not hybridize to a target sequence that isnon-complementary to the oligonucleotide at a single nucleotide residue(a single-nucleotide mismatch). Unfortunately, this type of singlenucleotide mismatch discrimination is possible only when fairly short(for example, <20 mer) oligonucleotides are used. The disadvantage ofusing such short oligonucleotides is that they hybridize weakly, even toa perfectly complementary sequence, and thus must be used underconditions of reduced stringency. If it were possible to achieve singlenucleotide mismatch discrimination under conditions of high stringency(such as those under which most amplification reactions are conducted),improvements in speed and efficiency would accrue in techniques such asallele-specific oligonucleotide hybridization, single nucleotidepolymorphism analysis, and functional genomics, to name just a few.

DISCLOSURE OF THE INVENTION

The present invention relates to a covalently bound oligonucleotide andminor groove binder combination which includes an oligonucleotide havinga plurality of nucleotide units, a 3′-end and a 5′-end, and a minorgroove binder moiety covalently attached to at least one of saidnucleotides. The minor groove binder is typically attached to theoligonucleotide through a linking group comprising a chain of no morethan 15 atoms. The minor groove binder moiety is a radical of a moleculehaving a molecular weight of approximately 150 to approximately 2000Daltons which molecule binds in a non-intercalating manner into theminor groove of double stranded DNA, RNA or hybrids thereof with anassociation constant greater than approximately 10³ M⁻¹.

In another aspect, the present invention relates to the process ofsynthesizing certain covalently bound oligonucleotide minor groovebinder combinations, and to the manner of using such combinations forhybridization probe and related analytical and diagnostic, as well astherapeutic (anti-sense and anti-gene) purposes.

It has now been discovered that conjugation of a minor groove binder(MGB) to an oligonucleotide (ODN) dramatically increases the stabilityof the hybrid formed between the oligonucleotide and its target.Increased stability (i.e., increased degree of hybridization) ismanifested in a higher melting temperature (T_(m): the temperature atwhich half of the base pairs have become unpaired) of hybrid duplexesformed by such MGB-oligonucleotide conjugates, compared to those formedby an unconjugated oligonucleotide of identical length and sequence.This effect is particularly pronounced for short oligonucleotides (e.g.,less than about 21 nucleotides in length) and makes possible, for thefirst time, the use of short oligonucleotides as probes and primers,under high stringency conditions. Conjugation of an oligonucleotide witha MGB, with its attendant increase in hybrid stability, does notadversely affect the ability of the conjugated oligonucleotide to serveas a primer. Therefore, it is now possible, using the methods andcompositions of the present invention, to use shorter oligonucleotidesthan previously required in techniques in which hybridization isrequired, such as polymerase chain reactions and hydrolyzable probeassays, which are generally conducted at high stringency, due to the useof high temperatures and thermophilic enzymes.

In addition to increased duplex stabilization, MGB-oligonucleotideconjugates retain the heightened sensitivity to sequence mismatch thatis characteristic of unconjugated short oligonucleotides with lowmelting temperatures. Thus, conjugation to a MGB endows very shortoligonucleotides (e.g. oligonucleotides containing less than about 21nucleotides) with greater specificity, by endowing them with thepotential to form hybrids having a stability characteristic of muchlonger oligonucleotides, while retaining the ability to discriminatebetween sequences differing by a single nucleotide. Use of shortoligonucleotides at high stringency now becomes possible, usingMGB-oligonucleotide conjugates.

The use of MGB-oligonucleotide conjugates as probes and primers providesimprovements in speed, sensitivity and versatility to a variety ofassays involving hybridization of oligonucleotides. Such assays arewell-known in the art and include, but are not limited to, singlenucleotide mismatch detection, in situ hybridization, polymerase chainreaction (PCR, see U.S. Pat. Nos. 4,683,202; 4,683,195 and 4,800,159),allele-specific oligonucleotide (ASO) hybridization (Huang et al. (1992)Acta Haematol. 88:92-95), detection of single-nucleotide polymorphism(Mullis and Faloona; Meth. Enzymol., vol. 155, Academic Press, New York,1987, pp. 335-50), microsatellite analysis using short tandem repeats(Tautz (1993) in “DNA Fingerprinting: State of the Science,” Pena etal., ed, Birkhauser, Basel, pp. 21-28), random amplification ofpolymorphisms in DNA (Williams et al, Meth. Enzymology, vol. 218,Academic Press, New York, 1993, pp. 704-740), DNA amplificationfingerprinting (Caetano-Anollés et al. (1991) Biotechnology 9:553-557),assays involving fluorescence energy transfer, assays involving releaseof label by exonuclease-mediated hydrolysis of a hybridizedoligonucleotide probe, assays involving ligation of two or moreoligonucleotides, etc.

All patents, patent applications and publications mentioned herein,either supra or infra, are hereby incorporated by reference in theirentirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the results of a slot blot hybridizationassay.

FIG. 2 shows the structure of1,2-dihydro-(3B)-pyrrolo[3,2-e]indole-7-carboxylate, also known asCDPI₃. Also shown are the structures of the linkers used for conjugationof CDPI₃ to the 5′ and 3′ ends of oligonucleotides.

FIG. 3 shows a comparison of unmodified and MGB-conjugated 16-mer,12-mer and 10-mer oligonucleotides as PCR primers. Conjugatedoligonucleotides contained a 5′-CDPI₃ moiety. The indicated pair ofprimers was used to amplify a segment of single-stranded M13mp19 DNAaccording to the procedure described in Example 1. PCR products wereanalyzed on 2% agarose gels stained with ethidium bromide.

In FIG. 3A, MGB-oligonucleotide conjugates (lanes 14) were compared tounmodified oligonucleotides (lanes 5-8) as reverse primers. In allcases, the oligonucleotides were 16-mers, and the annealing temperaturewas 45° C. The specific pairs of oligonucleotides used as primers, andthe predicted sizes of the products, were as follows. Lane 1: 4-C and 1(307 nucleotides). Lane 2: 9-C and 1 (297 nucleotides). Lane 3: 12-C and1 (217 nucleotides). Lane 4: 13-C and 1 (181 nucleotides). Lane 5: 4 and1 (307 nucleotides). Lane 6: 9 and 1 (297 nucleotides). Lane 7: 12 and 1(217 nucleotides). Lane 8: 13 and 1 (181 nucleotides); See Table 6 forthe sequences and structures of the oligonucleotides, and for theirlocation within the M13mp19 genome. Lane M comprises molecular weightmarkers, whose size (in nucleotides) is given to the left of the Figure.

In FIG. 3B, MGB-oligonucleotide conjugates (lanes 14) were compared tounmodified oligonucleotides (lanes 5-8) as reverse primers. In allcases, the oligonucleotides were 16-mers, and the annealing temperaturewas 68° C. The specific pairs of oligonucleotides used as primers, andthe predicted sizes of the products, were as follows. Lane 1: 4-C and 1(307 nucleotides). Lane 2: 9-C and 1 (297 nucleotides). Lane 3: 12-C and1 (217 nucleotides). Lane 4: 13-C and 1 (181 nucleotides). Lane 5: 4 and1 (307 nucleotides). Lane 6: 9 and 1 (297 nucleotides). Lane 7: 12 and 1(217 nucleotides). Lane 8: 13 and 1 (181 nucleotides). See Table 6 forthe sequences and structures of the oligonucleotides, and for theirlocation within the M13mp19 genome. Lane M comprises molecular weightmarkers as in FIG. 3A.

In FIG. 3C, 10-mer (lane 1; oligonucleotides 3-C and 7-C) and 12-mer(lane 2; oligonucleotides 6-C and 2-C) MGB-oligonucleotide conjugateswere used as primers and the annealing temperature was 55° C. Thepredicted product length was 307 nucleotides for both lanes. Lane Mcomprises molecular weight markers as in FIG. 3A.

FIG. 4 shows a comparison of unmodified and MGB-conjugated 8-mer and6-mer oligonucleotides as PCR primers. Conjugated oligonucleotidescontained a 5′-CDPI₃ moiety. The indicated pair of primers were used toamplify a segment of single-stranded M13mp19 DNA using a touchdown PCRprotocol as described in Example 1. Products were analyzed on 8%polyacrylamide sequencing gels and visualized by silver staining.

In FIG. 4A, the forward primer was a 10-mer, the reverse primers were8-mers, and the annealing temperature was gradually decreased from 55 to41° C. The specific primer pairs, and the predicted sizes of theproducts, were as follows. Lane 1: 11 and 3 (217 nucleotides). Lane 2: 8and 3 (297 nucleotides). Lane 3: 11-C and 3-C (217 nucleotides). Lane 4:8-C and 3-C (297 nucleotides). Lane M denotes molecular weight markersfrom a Hae III digest of ΦX174 DNA, whose sizes (in nucleotides) aregiven to the right of the figure.

In FIG. 4B, the forward primer was a 10-mer, the reverse primers were6-mers, and the annealing temperature was gradually decreased from 50 to37° C. The specific primer pairs, and the predicted sizes of theproducts, were as follows. Lane 1: 10-C and 3-C (295 nucleotides). Lane2: 5-C and 3-C (305 nucleotides). Lane M denotes molecular weightmarkers from a Hae III digest of ΦX174 DNA, as in FIG. 4A.

FIG. 5 shows the nucleotide sequence of the E. coli supF gene containedin the plasmid pSP189 (SEQ ID No.: 40), indicating the locations of thetarget sequences for the amplification primers (labeled “Primer 1” and“Primer 2”), the region that served as target in a hydrolyzable probeassay (labeled “15-mer”), and the single-nucleotide substitutions thatwere introduced into the target sequence for the experiment shown inFIG. 6 (shown underneath the region labeled “15-mer”).

FIG. 6 shows results of a hydrolyzable probe assay, using MGB-conjugated15-mer probes wherein all guanine bases in the probe were substitutedwith the guanine analogue ppG. The target was the E. coli supF gene.Annealing/elongation was conducted at 75° C. for 20 sec per cycle.

MODES FOR CARRYING OUT THE INVENTION

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques in organic chemistry, biochemistry,oligonucleotide synthesis and modification, nucleic acid hybridization,molecular biology, microbiology, genetics, recombinant DNA; and relatedfields as are within the skill of the art. These techniques are fullyexplained in the literature. See, for example, Maniatis, Fritsch &Sambrook, MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring HarborLaboratory Press (1982); Sambrook et al., supra; Ausubel, et al.,CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons (1987, 1988,1989, 1990, 1991, 1992, 1993, 1994, 1995, 1996); Gait (ed.),OLIGONUCLEOTIDE SYNTHESIS: A PRACTICAL APPROACH, IRL Press (1984);Eckstein (ed.), OLIGONUCLEOTIES AND ANALOGUES: A PRACTICAL APPROACH, IRLPress (1991).

A prominent feature of the novel composition of matter of the presentinvention is that a minor groove binding molecule is covalently bound toan oligonucleotide. As is noted in the introductory section of thepresent application for patent, a minor groove binder is a molecule thatbinds within the minor groove of double stranded deoxyribonucleic acid(DNA). Although a general chemical formula for all known minor groovebinding compounds cannot be provided because such compounds have widelyvarying chemical structures, compounds which are capable of binding inthe minor groove of DNA, generally speaking, have a crescent shape threedimensional structure. Most minor groove binding compounds of the priorart have a strong preference for A-T (adenine and thymine) rich regionsof the B form of double stranded DNA. The minor groove bindingcompounds, or more accurately stated moieties of theoligonucleotide-minor groove binding conjugates of the presentinvention, also have the same preference. (The oligonucleotide-minorgroove binding conjugates of the present invention are hereinaftersometimes referred to as ODN-MGB.) Nevertheless, minor groove bindingcompounds which would show preference to C-G (cytosine and guanine) richregions are also theoretically possible. Therefore, ODN-MGB compoundsincorporating a radical or moiety derived from minor groove bindermolecules having preference for C-G regions are also within the scope ofthe present invention. The preference for A-T regions of the known minorgroove binders is currently explained by the existence of an unfavorablesteric interference between the 2-amino group of guanine and some wellknown minor groove binders. However, as it will become apparent from theensuing further description, when guanine is replaced by hypoxanthine inan ODN-MGB of the present invention, the potential for the above-notedunfavorable steric interference no longer exists and strong binding ofthe ODN-MGB to a complementary strand may occur.

Generally speaking, minor groove binding compounds known in the priorart do not bind to double stranded RNA or to a double stranded hybrid ofDNA and RNA. However, the ODN-MGB compounds of the present inventionexhibit potential for binding to single stranded RNA, and the foregoingfeature forms another interesting and novel aspect of the presentinvention.

Examples of known minor groove binding compounds of the prior art, whichcan, in accordance with the present invention, be covalently bound toODNs to form the novel ODN-MGB conjugates are certain naturallyoccurring compounds such as netropsin, distamycin and lexitropsin,mithramycin, chromomycin A₃, olivomycin, anthramycin, sibiromycin, aswell as further related antibiotics and synthetic derivatives. Certainbisquarternary ammonium heterocyclic compounds, diarylamidines such aspentamidine, stilbamidine and berenil, CC-1065 and related pyrroloindoleand indole polypeptides, Hoechst 33258, 4′-6-diamidino-2-phenylindole(DAPI) as well as a number of oligopeptides consisting of naturallyoccurring or synthetic amino acids are minor groove binder compounds.The chemical structures of the following examples are illustrated below.

For the purposes of the present invention a molecule is a minor groovebinder if it is capable of binding within the minor groove of doublestranded DNA with an association constant of 10³ M⁻¹ or greater. Thistype of binding can be detected by well established spectrophotometricmethods, such as ultraviolet (u.v.) and nuclear magnetic resonance (nmr)spectroscopy and also by gel electrophoresis. Shifts in u.v. spectraupon binding of a minor groove binder molecule, and nmr spectroscopyutilizing the “Nuclear Overhauser” (NOSEY) effect are particularly wellknown and useful techniques for this purpose. Gel electrophoresisdetects binding of a minor groove binder to double stranded DNA orfragment thereof, because upon such binding the mobility of the doublestranded DNA changes.

Intercalating molecules or agents are readily distinguished from minorgroove binders on the basis that the intercalating agents are flataromatic (preferably polycyclic) molecules versus the “crescent shape”or analogous geometry of the minor groove binders. An experimentaldistinction can also be made by nmr spectroscopy utilizing the NuclearOverhauser effect.

As noted above, for the purposes of the present invention a molecule isa minor groove binder if its association constant within the minorgroove of double stranded DNA is 10³ M⁻¹ or greater. However, some minorgroove binders bind to the high affinity sites of double stranded DNAwith an association constant of the magnitude of 10⁷ to 10⁹ M⁻¹.

In accordance with the present invention, the minor groove bindermolecule is derivatized, in essence formed into a “radical” and linkedto an appropriate covalent structure or chain of atoms that attaches theminor groove binder to the ODN. In a sense, the linking “chain” can andsometimes is considered as part of the minor groove binder since thenature of the linkage is such that it does not adversely affect theminor groove binding properties of the ODN-MGB molecule. However, itsuits the present description better to conceptually separate the minorgroove binder from the group that covalently attaches it to the ODN. Theradical “formed” from the minor group binder molecule is hereinafterreferred to as the “minor groove binder moiety”, and the covalentlinkage (which may be a chain of up to approximately 15 atoms) thatattaches the minor groove binder moiety to the oligonucleotide is calledthe “linking group”. The preferred embodiments of the minor groovemoieties in accordance with the present invention are described indetail after description of the oligonucleotide portion of the ODN-MGBconjugate compounds of the present invention.

Broadly speaking, the oligonucleotide portion of the ODN-MGB conjugatesof the present invention comprise approximately 3 to 100 nucleotideunits. The nucleotide units which can be incorporated into the ODNs inaccordance with the present invention include the major heterocyclicbases naturally found in nucleic acids (uracil, cytosine, thymine,adenine and guanine) as well as naturally occurring and syntheticmodifications and analogs of these bases such as hypoxanthine,2-aminoadenine, 2-thiouracil, 2-thiothymine, 5-N⁴ ethenocytosine,4-aminopyrrazolo[3,4-d]pyrimidine and6-amino-4-hydroxy-[3,4-d]pyrimidine. The respective structures of the2-deoxyribosides of 5-N⁴ ethenocytosine4-aminopyrrazolo[3,4-d]pyrimidine and of6-amino-4-hydroxy-[3,4-d]pyrimidine are shown below.

In addition, the nucleotide units which are incorporated into the ODNsof the ODN-MGB conjugates of the present invention may have across-linking function (an alkylating agent) covalently bound to one ormore of the bases, through a linking arm. Since the ODN-MGB conjugateshaving an attached cross-linking agent form an important class ofpreferred embodiments of the present invention these structures will bedescribed in more detail below.

The “sugar” or glycoside portion of the ODN-MGBs of the presentinvention may comprise deoxyribose, ribose, 2-fluororibose, 2-O alkyl oralkenylribose where the alkyl group may have 1 to 6 carbons and thealkenyl group 2 to 6 carbons. In the naturally occurring nucleotides andin the herein described modifications and analogs the deoxyribose orribose moiety forms a furanose ring, the glycosydic linkage is of the βconfiguration and the purine bases are attached to the sugar moiety viathe 9-position, the pyrimidines via the 1-position and thepyrazolopyrimidines via the 1-position. Presently,oligodeoxyribonucleotides are preferred in accordance with the presentinvention, therefore the preferred sugar is 2-deoxyribose. Thenucleotide units of the ODN's are interconnected by a “phosphate”backbone, as is well known in the art. The ODNs of the ODN-MGBconjugates of the present invention may include, in addition to the“natural” phosphodiester linkages, phosphorothiotes andmethylphosphonates.

The ODNs of the ODN-MGB conjugates of the present invention may alsohave a relatively low molecular weight “tail moiety” attached to eitherat the 3′- or 5′-end. The “tail moiety” in this particular context is tobe distinguished from the minor groove binding moiety, which ispreferably also attached to the 3′ or 5′ ends, or to both. Thus, in thiscontext the “tail moiety” if present at all, is attached to the end ofthe ODN which does not bear the minor groove binder moiety. By way ofexample, a tail molecule may be a phosphate, a phosphate ester, an alkylgroup, and aminoalkyl group, or a lipophilic group.

With regard to the possible variations of the nucleotide units, the“phosphate backbone” and “tail” of the ODNs of the ODN-MGB conjugates ofthe present invention, the following should be kept in mind. Theprincipal useful action of the ODN-MGB conjugates of the presentinvention lies in the ability of the ODN portion of the molecule to bindto a complementary sequence in single stranded DNA, RNA, double strandedDNA, and DNA-RNA hybrid, in a manner in which the minor groove bindingmoiety is incorporated in the newly formed “duplex” and therebystrengthens the bond, that is, increases the melting temperature (andassociation constant) of the newly formed duplex. Additionally, thosepreferred embodiments of the ODN-MGB conjugates of the present inventionwhich include a cross-linking agent, also result in permanent covalentattachment of the ODN-MGB molecule to the complementary DNA or RNAstrand, resulting in a permanently bound form. In light of theforegoing, those skilled in the art will readily understand that theprimary structural limitation of the various component parts of the ODNportion of the ODB-MGB conjugate of the present invention lies only inthe ability of the ODN portion to form a complementary strand to anyspecific target sequence, and that a large number of structuralmodifications, per se known in the art, are possible within thesebounds. Moreover, synthetic methods for preparing the variousheterocyclic bases, nucleosides, nucleotides and oligonucleotides whichcan form the ODN portion of the ODN-MGB conjugates of the presentinvention, are generally speaking well developed and known in the art.N₄,N₄-ethano-5-methyldeoxycytidine, its nucleoside, nucleotide and/oroligonucleotides incorporating this base can be made in accordance withthe teachings of Webb, T. R.; Matteucci, M. D. Nucleic Acids Res., 1986,14, 7661-7674, Webb, T. R.; Matteucci, M. D. J. Am. Chem. Soc., 1986,108, 2764. 4-aminopyrazolo[3,4-d]pyrimidine,6-amino-4-hydroxypyrazolo[3,4-d]pyrimidine, their nucleosides,nucleotides and oligonucleotides incorporating this base can be made inaccordance with the teachings of Kazimierczuk et al. J. Am. Chem. Soc.,1984, 106, 6379-6382. Whereas oligonucleotide synthesis, in order toprepare an ODN of specific predetermined sequence so as to becomplementary to a target sequence, can be conducted in accordance withthe state of the art, a preferred method is described below. Thepreferred method incorporates the teaching of U.S. Pat. No. 5,419,966,the disclosure of which is expressly incorporated herein by reference.

The linking group is a moiety which covalently links the ODN portion ofthe conjugate to the minor groove binder moiety. Preferably, the linkinggroup is such that the linkage occurs through a chain of no more than 15atoms. Also preferably in accordance with the present invention theminor groove binder moiety is covalently attached to either the 3′ or 5′end of the oligonucleotide. Nevertheless, attachment to a nucleotide inintermediate position, and particularly to the heterocyclic base of thenucleotide in intermediate position is also within the scope of theinvention. Generally speaking, the linking group is derived from abifunctional molecule so that one functionality such as an aminefunctionality is attached for example to the phosphate on the 5′ end ofthe ODN, and the other functionality such as a carbonyl group (CO) isattached to an amino group of the minor groove binder moiety.Alternatively, the linking group may be derived from an amino alcohol sothat the alcohol function is linked, for example, to the 3′-phosphateend of the ODN and the amino function is linked to a carbonyl group ofthe minor groove binder moiety. Still another alternative of a linkinggroup includes an aminoalcohol (attached to the 3′-phosphate with anester linkage) linked to an aminocarboxylic acid which in turn is linkedin a peptide bond to the carbonyl group of the minor groove binder.Thus, preferred embodiments of the linking group have the formulas—HN(CH₂)_(m)CO, O(CH₂)_(m)CO and(CH₂)_(m)CH(OH)_(m)(CH₂)_(m)NHCO(CH₂)_(m)NH where the limitation on m isthat the minor groove binder moiety should not be separated by more thanapproximately 15 atoms from the ODN. Preferred embodiments of linkinggroups are —O(CH₂)₆NH, —OCH₂CH(OH)CH₂NHCOCH₂CH₂NH and —HN(CH₂)₅CO. As itwas noted above, the linking group could also be conceptualized as partof the minor groove binder moiety, which in that case would beconsidered directly attached to the ODN.

The basic limitation for the minor groove binder moiety has been setforth above, and is not definable by specific chemical structure. Inaddition to the molecular structure which causes minor groove binding,the minor groove binder moiety may also carry additional functions, aslong as those functions do not interfere with minor groove bindingability. For example a reporter group, which makes the minor groovebinder readily detectable by color, uv. spectrum or other readilydiscernible physical or chemical characteristic, may be covalentlyattached to the minor groove binder moiety. An example for such areporter group is a diazobenzene function which in the example of apreferred embodiment is attached to a carbonyl function of the minorgroove binder through a —HN(CH₂)_(m)COO(CH₂)_(m)S(CH₂)_(m)— bridge.Again, the reporter group or other like function carried by the minorgroove binder can also be conceptualized as part of the minor groovebinder moiety itself.

Preferred embodiments of the ODN-MGB conjugates are defined by thefollowing chemical Formula 1. This definition includes the preferredembodiments of the minor groove binder moiety in accordance with thepresent invention, which may also include all or part of the linkinggroup and other appendant groups such as a reporter group, as discussedabove:

where x is O or S;

q is an integer between 3 to 100;

R₈ is H, OH, alkoxy having 1 to 6 carbons, O—C₂-C₆alkenyl, or F;

B is an aglycon selected from a group consisting of a heterocyclic basenaturally found in nucleic acids and hypoxanthine, 2-aminoadenine,2-thiouracil, 2-thiothymine, 5-N ⁴-ethenocytosine,4-aminopyrrazolo[3,4-d]pyrimidine, 6-amino-4-hydroxy-[3,4-d]pyrimidine;

W₁ is H, PO(OH)₂ or a salt thereof, or a minor groove binder moietyattached to the 3′ or 5′ end of said oligonucleotide, the W₁ groupincluding the linking group which covalently binds the minor groovebinder moiety to the oligonucleotide through no more than 15 atoms;

W₂ is absent or is a minor groove binder moiety attached to one of theaglycons B, the W₂ group including the linking group which covalentlybinds the minor groove binder moiety to said aglycon, or W₂ is across-linking functionality including a linker arm which covalentlybinds the cross-linking functionality to said aglycon, wherein the minorgroove binder moiety is a radical of a molecule having a molecularweight of approximately 150 to approximately 2000 Daltons that bind in anon-intercalataing manner into the minor groove of double stranded DNA,RNA or hybrids thereof with an association constant greater thanapproximately 10³, with the proviso that at least one of said W₁ and W₂groups is a minor groove binder moiety; and

wherein further the minor groove binder moiety including the linkinggroup has the formula selected from the group consisting of groups (a),(b), (c), (d) and (e):

R₁—(HN—Y₁—CO)_(n)—R₂  (a)

where Y₁ represents a 5-membered ring having two double bonds and 0 to3′ heteroatoms selected from the group consisting of N, S and O, the NHand CO groups are attached respectively to two ring carbons which areseparated by one ring atom from one another, the ring atom positionedbetween said two ring carbons is substituted only with H or isunsubstituted, each of the remaining ring atoms may be optionallysubstituted with 1; 2 or 3 R₃ groups;

R₁—(R₆N—Y₂—CO)_(n)—R₂  (b)

here Y2 is a ring system consisting of a 6-membered aromatic ringcondensed with a 5-membered ring having one double bond, the condensedring system having 0 to 3 heteroatoms selected from the group consistingof N, S and O, each of the R₆N and CO groups is attached to a ringcarbon which is in a different ring of the condensed ring system, andwhich is the second ring atom, respectively, from one common bridge headring atom, the CO and NR₆ groups thereby positioning 2 non-bridgeheadring atoms between themselves on one side and 3 non-bridgehead ringatoms on the other side of the condensed ring system, the twonon-bridgehead ring atoms on the one side being optionally substitutedwith an R₇ group, the three non-bridgehead ring atoms on the other sideof the condensed ring system being optionally substituted with an R₃group;

R1-(CO—Y₃—NH)n-R  (c)

where Y₃ is a 6-membered aromatic ring having 0 to 3 N heteroatoms, andwhere each of the CO and NH groups is attached to a ring carbon, saidring carbons being in 1,4 position relative to one another, two ringatoms not occupied by the CO or NH groups on either one of the two sidesof the 6-membered ring being optionally substituted with an R₃ group,the two ring atoms not occupied on the other side of the 6 membered ringbeing optionally substituted with an R₇ group;

R₁—(HN—Y₄—HN—CO—Y₄—CO)_(p)—R₂  (d)

where Y₄ is a 6-membered aromatic ring having 0 to 3 N heteroatoms, andwhere each of the CO and NH groups is attached to a ring carbon, saidring carbons being in 1,4 position relative to one another in each ring,two ring atoms not occupied by the CO or NH groups on either one of thetwo sides of the 6-membered ring being optionally substituted with an R₃group, the two ring atoms not occupied on the other side of the 6membered ring being optionally substituted with an R₇ group;

R₁—(Y₅)_(n)—R₂  (a)

where Y₅ is a ring system consisting of a 6 membered aromatic ringcondensed with a 5-membered ring having one double bond, the condensedring system having 0 to 3 heteroatoms selected from the group consistingof N, S and O, each of the R₁ and R₂ groups is attached to a ring carbonwhich is in a different ring of the condensed ring system, and which isthe second ring atom, respectively, from one common bridgehead ringatom, the R₁ and R₂ groups thereby positioning 2 non-bridgehead ringatoms between themselves on one side and 3 non-bridgehead ring atoms onthe other side of the condensed ring system, the two non-bridgehead ringatoms on the one side being optionally-substituted with an R₇ group, thethree non-bridgehead ring atoms on the other side of the condensed ringsystem being optionally substituted with an R₃ group;

where R₁ and R₂ independently are H, F, Cl, Br, I, NH₂, NHR₄, N(R₄)₂,N(R₄)₃ ⁺, OH, —O—, —S—, OR₄, SH, SR₄, COR₄, CONHR₄, CON(R₄)₂, R₄,H₂N(CH₂)_(m)CO, CONH₂, CONHR₄,H₂N(CH₂)_(m)COO(CH₂)_(m)S(CH₂)_(m)C₆H₄NNC₆H₄, —HN(CH₂)_(m)CO, —CONH—,—CONR₄, HN(CH₂)_(m)COO(CH₂)_(m)S(CH₂)_(m)C₆H₄NNC₆H₄, and—(CH₂)_(m)CH(OH)(CH₂)_(m)NHCO(CH₂)_(m)NH—, or one of the R₁ and R₂groups is absent;

R₃ is selected from the group consisting of F, Cl, Br, I, NH₂, NHR₄,N(R₄)₂, N(R₄)₃ ⁺, OH, OR₄, SH, SR₄, COR₄, CONH₄, CON(R₄)₂ and R₄, or theR₃ groups may form a 3, 4, 5 or 6 membered ring condensed to the Y₁ring;

R₄ is an alkyl or cycloalkyl group having 1 to 20 carbons, an alkenyl orcycloalkenyl group having 1 to 20 carbons and 1 to 3 double bonds, acarbocyclic aromatic group of no more than 25 carbons, a heterocyclicaromatic group of no more than 25 carbons, a carbocyclic or heterocyclicarylalkyl group of no more than 25 carbons, where R₄ may be optionallysubstituted with 1, 2 or 3 F, Cl, Br, I, NH₂, NHR₅, N(R₅)₂, N(R₅)₃ ⁺,OH, OR₅, SH, SR₅, COR₅, CONHR₅, CON(R₅)₂ or R₅ groups;

R₅ is alkyl of 1 to 6 carbons,

R₆ is H, alkyl of 1 to 5 carbons, or R₆ and R₇ jointly form a 4,5, or 6membered ring, optionally an —O—, —S—, —NH—, NCH₃—, or N-lower alkylgroup being part of said ring;

R₇ is F, methyl or ethyl; —CH₂—, or —CH₂CH₂—;

m is an integer between 1 to 10;

n is an integer between 1 to 10, and

p is an integer between 1 to 5.

Still more preferred embodiments of the ODN-MBG conjugates of thepresent invention are those where the minor groove binder moiety isdefined as follows:

(1) the minor groove binding moiety is represented by formula (a) aboveand the five membered ring has the structure

(2) the minor groove binding moiety is represented by formula (a) abovewherein the five membered ring has the structure

and

(3) the minor groove binding moiety is represented by formula (b) andthe condensed ring system has the structure

Embodiments Containing a Crosslinking Functionality

A class of preferred embodiments of the ODN-MGB conjugates of thepresent invention also include one or more cross-linking functionalitieswhereby after the ODN-MGB conjugate is bound to a complementary targetsequence of DNA, RNA or fragment thereof, the crosslinking functionalityirreversibly reacts with the target and forms a covalent bond therewith.Advantages of such covalent linking to a target sequence are inanalytical, diagnostic use, as in hybridization probes, and intherapeutic (anti-sense and anti-gene) applications. The minor groovebinder moiety which is also covalently bound to the ODN that complementsthe target sequence, enhances the initial non-covalent binding of theODN-MGB conjugate to the target sequence and therefore facilitates thesubsequent covalent bonding through the cross-linking function. Thefollowing considerations are pertinent as far as the cross-linkingfunctionalities or agents incorporated into this class of ODN-MGBconjugates are concerned.

The cross-linking agents incorporated in the present invention arecovalently bonded to a site on the ODN-MGB. Its length and stericorientation should be such that it can reach a suitable reaction site inthe target DNA or RNA sequence after the ODN-MGB is hybridized with thetarget. By definition, the crosslinking functionality or agent has areactive group which will react with a reactive group of the target DNAor RNA sequence. The cross-linking agent (or agents) may be covalentlyattached to one or more of the heterocyclic bases, to the sugar ormodified sugar residues, or to the phosphate or modified phosphatefunctions of the ODN-MGB conjugates. The cross-linking agent may also beattached to the minor groove binder moiety as long as it does notinterfere with its minor groove binding ability. Preferably thecross-linking agent or functionality is attached to one of theheterocyclic bases.

In simple terms the cross-linking agent itself may conceptually bedivided into two groups or moieties, namely the reactive group, which istypically and preferably an electrophilic leaving group (L), and an“arms” (A) which attaches the leaving group L to the respective site onthe ODN-MGB. The leaving group L may be chosen from, for example, suchgroups as chloro, bromo, iodo, SO₂R′″, or S⁺R′″R″″, where each of R′″and R′″ is independently C₁₋₆alkyl or aryl or R′″ and R″″ together forma C₁₋₆alkylene bridge. Chloro, bromo and iodo are preferred. Withinthese groups haloacetyl groups such as —COCH₂I, and bifunctional“nitrogen mustards”, such as —N—[(CH₂)₂—Cl]₂ are preferred. The leavinggroup will be altered by its leaving ability. Depending on the natureand reactivity of the particular leaving group, the group to be used ischosen in each case to give the desired specificity of the irreversiblybinding probes.

Although as noted above the “arm” (or linker arm) A may conceptually beregarded as a single entity which covalently bonds the ODN-MGB to theleaving group L, and maintains the leaving group L at a desired distanceand steric position relative to the ODN-MGB, in practice the “arm” A maybe constructed in a synthetic scheme where a bifunctional molecule iscovalently linked to the ODN-MGB, or to the ODN before the minor groovebinder moiety is attached (for example by a phosphate ester bond to the3′ or 5′ terminus, by a carbontocarbon bond to a heterocyclic base or bycarbon to nitrogen bond to an amino substituted heterocyclic base)through its first functionality, and is also covalently linked throughits second functionality (for example an amine) to a “hydrocarbylbridge” (alkyl bridge, alkylaryl bridge or aryl bridge, or the like)which, in turn, carries the leaving group L.

A general formula of the cross linking function is thus -A-L, or -A-L₂where L is the above defined leaving group and A is a moiety that iscovalently linked to the ODN-MGB. The A “arm” moiety itself should beunreactive (other than through the leaving group L) under the conditionsof hybridization of the ODN-MGB with the target sequence, and shouldmaintain the leaving group L in a desired steric position and distancefrom the desired site of reactions such as an N-7 position of aguanosine residue in the target sequence. Generally speaking, the lengthof the A group should be equivalent to the length of a normal alkylchain of approximately 2 to 20 carbons.

An exemplary more specific formula for a class of preferred embodimentsof the cross-linking function is

—(CH₂)_(q)—Y—(CH₂)_(m)-L

where L is the leaving group, defined above, each of m and q isindependently 0 to 8, inclusive, and where Y is defined as a “functionallinking group”. For clarity of description this “functional linkinggroup” is to be distinguished from the “linking group” that attaches theminor groove binder moiety to the ODN, although the functional linkinggroups described here for attaching the cross-linking agent can also beused for attaching a minor groove binder moiety to either end of theODN, or to a nucleotide in intermediate position of the ODN. A“functional linking group” is a group that has two functionalities, forexample —NH₂ and —OH, or —COOH and —OH, or —COOH and —NH₂, which arecapable of linking the (CH₂)_(q) and (CH₂) bridges. An acetylenicterminus (HC═C—) is also a suitable functionality for Y, because it canbe coupled to certain heterocycles, as described below.

other exemplary and more specific formulas for a class of preferredembodiments of the cross-linking function are

—(CH₂)_(q)—NH—CO—(CH₂)_(m)—(X)_(n)—N(R₁)—(CH₂)_(p)-L

and

—(CH₂)_(q′)—O—(CH₂)_(q″)—NH—CO—(CH₂)_(m)—(X)_(n)—N(R₁)—(CH₂)_(p)-L

where q, m and L are defined as above in connection with the descriptionof the cross-linking functions, q′ is 3 to 7 inclusive, q″ is 1 to 7inclusive, x is phenyl or simple substituted phenyl (such as chloro,bromo, lower alkyl or lower alkoxy substituted phenyl), n is 0 or 1, pis an integer from 1 to 6, and R₁ is H, lower alkyl or (CH₂)_(p)-L.Preferably p is 2. Those skilled in the art will recognize that thestructure —N(R₁)—(CH₂)₂-L describes a “nitrogen mustard”, which is aclass of potent alkylating agents. Particularly preferred are withinthis class of ODN-MGB conjugates those where the crosslinking agentincludes the functionality N(R₁)—(CH₂)₂-L where L is halogen, preferablychlorine; and even more preferred are those ODN-MGB conjugates where thecross-linking agent includes the grouping N—[(CH₂)₂-L]₂ (a“bifunctional” N-mustard).

A particularly preferred partial structure of the cross-linking agentincludes the grouping

—O—(CH₂)₃—C₆H₄—N—[(CH₂)₂Cl]₂.

In a preferred embodiment the just-noted cross-linking group is attachedto an n-hexylamine bearing tail at the 5′ and 3′ ends of the ODN inaccordance with the following structure:

R′—O—(CH₂)₆—NH—CO—(CH₂)₃—C₆H₄—N—[(CH₂)₂Cl]₂

where R′ signifies the terminal 5′ or 3′-phosphate group of the ODN. Theother terminal, or a nucleotide in an intermediate position bears theminor groove binder moiety.

In accordance with other preferred embodiments, the cross-linkingfunctionality is covalently linked to the heterocyclic base, for exampleto the uracil moiety of a 2′-deoxyuridylic acid building block of theODN-MGB conjugate. The linkage can occur through the intermediacy of anamino group, that is, the “armleaving group combination” (A-L) may beattached to a 5-amino-2′-deoxyuridylic acid building unit of the ODN. Instill other preferred embodiments the “arm-leaving group combination”(A-L) is attached to the 5-position of the 2′-deoxyuridylic acidbuilding unit of the ODN by a carbon-to-carbon bond. Generally speaking,5-substituted-2′-deoxyuridines can be obtained by an adaptation of thegeneral procedure of Robins et al. (Can. J. Chem., 60:554 (1982); J.Org. Chem., 48:1854 (1983)). In accordance with this adaptation,palladium-mediated coupling of a substituted 1-alkyne to5-iodo-2′-deoxyuridine gives an acetylene-coupled product. Theacetylenic dUrd analog is reduced, with Raney nickel for example, togive the saturated compound, which is then used for direct conversion toa reagent for use on an automated DNA synthesizer. Examples of reagentswhich can be coupled to 5-iodo-2′-deoxyuridine in accordance with thismethod are

HC≡CCH₂OCH₂CH₂N(CO)₂C₆H₄ (phtalimidoethoxypropyne) andHC≡CCH₂0CH₂CH₂NHCOCF₃ (trifluoroacetamidoethoxypropyne).

In these examples the nucleosides which are obtained in this scheme areincorporated into the desired ODN; and the alkylating portion of thecrosslinking agent is attached to the terminal amino group only afterremoval of the respective phtalic or trifluoroacetyl blocking groups,other examples of nucleotides where the crosslinking agent is attachedto a heterocyclic base, are 2′-deoxy-4-aminopyrazolo[3,4-d]pyrimidinederivatives. These compounds can be made in accordance with the teachingof published PCT application WO: 90/03370 (published on Apr. 5, 1990).

Discussing still in general terms the structures of the modified ODNs ofthe present invention, it is noted that examination of double-strandedDNA by balland-stick models and high resolution computer graphicsindicates that the 7-position of the purines and the 5-position of thepyrimidines lie in the major groove of the B-form duplex ofdouble-stranded nucleic acids. These positions can be substituted withside chains of considerable bulk without interfering with thehybridization properties of the bases. These side arms may be introducedeither by derivatization of dThd or dCyd, or by straightforward totalsynthesis of the heterocyclic base, followed by glycosylation. Thesemodified nucleosides may be converted into the appropriate activatednucleotides for incorporation into oligonucleotides with an automatedDNA synthesizer. With the pyrazolo[3,4-d]pyrimidines, which are analogsof adenine, the crosslinking arm is attached at the 3-position, which isequivalent to the 7-position of purine.

The crosslinking side chain (arm=A) should be of sufficient length toreach across the major groove from a purine 7- or 8-position, pyrimidine5-position, pyrrolopyrimidine 5-position or pyrazolopyrimidine3-position and reacting with the N-7 of a purine (preferably guanine)located above (on the oligomer 3′side) the base pair containing themodified analog. The crosslinking side chain (arm=A) holds thefunctional group away from the base when the base is paired with anotherwithin the double-stranded complex. As noted above, broadly the arm Ashould be equivalent in length to a normal alkyl chain of 2 to 20carbons. Preferably, the arms include alkylene groups of 1 to 12 carbonatoms, alkenylene groups of 2 to 12 carbon atoms and 1 or 2 olefinicbonds, alkynylene groups of 2 to 12 carbon atoms and 1 or 2 acetylenicbonds, or such groups substituted at a terminal point with nucleophilicgroups such as oxy, thio, amino or chemically blocked derivativesthereof (e.g., trifluoroacetamido, phthalimido, CONR′, NR′CO, andS0₂NR′, where R′═H or C₁₋₆alkyl). Such functionalities, includingaliphatic or aromatic amines, exhibit nucleophilic properties and arecapable of serving as a point of attachment to such groups as

—(CH₂)_(m)-L, and

—CO—(CH₂)_(m)—(X)_(n)—N(R₁)—(CH₂)_(p)-L

which are described above as components of exemplary cross-linkingfunctional groups.

After the nucleoside or nucleotide unit which carries the crosslinkingfunctionality A-L, or a suitable precursor thereof, (such as the—(CH₂)_(q)—NH₂ or —(CH₂)_(q)—Y group, where Y terminates with anucleophilic group such as NH₂) is prepared, further preparation of themodified oligonucleotides of the present invention can proceed inaccordance with state of the art. Thus, to prepare oligonucleotides,protective groups are introduced onto the nucleosides or nucleotides andthe compounds are activated for use in the synthesis ofoligonucleotides. The conversion to protected, activated forms mayfollow the procedures as described for 2′-deoxynucleosides in detail inseveral reviews. See, Sonveaux, Bioorganic Chemistry, 14:274-325 (1986);Jones, in “Oligonucleotide Synthesis, a Practical Approach”, M. J. Gait,Ed., IRL Press, p. 23-34 (1984).

The activated nucleotides are incorporated into oligonucleotides in amanner analogous to that for DNA and RNA nucleotides, in that thecorrect nucleotides will be sequentially linked to form a chain ofnucleotides which is complementary to a sequence of nucleotides intarget DNA or RNA. The nucleotides may be incorporated eitherenzymatically or via chemical synthesis. The nucleotides may beconverted to their5′-O-dimethoxytrityl-3′-(N,N-diisopropyl)phosphoramidite cyanoethylester derivatives, and incorporated into synthetic oligonucleotidesfollowing the procedures in “Oligonucleotide Synthesis: A PracticalApproach”, supra. The N-protecting groups are then removed, along withthe other oligonucleotide blocking groups, by post-synthesis aminolysis,by procedures generally known in the art.

In a preferred embodiment, the activated nucleotides may be useddirectly on an automated DNA synthesizer according to the procedures andinstructions of the particular synthesizer employed. Theoligonucleotides may be prepared on the synthesizer using the standardcommercial phosphoramidite or H-phosphonate chemistries.

A moiety containing the leaving group, such as a haloacyl group, or—CO—(CH₂)_(m)—(X)_(n)—N(R₁)—(CH₂)_(p)-L group (even more preferably aCO—(CH₂)₃—C₆H₄—N—[CH₂CH₂Cl]₂) may be added to the aminoalkyl or liketails (—CH₂)_(q)—Y) following incorporation into oligonucleotides andremoval of any blocking groups.

In the situations where the cross linking agent (A-L moiety) is attachedto the 3′ or 5′ terminus of the oligonucleotide, for example by analkylamine linkage of the formula —(CH₂)_(g)—Y (Y terminating in anamine), the oligonucleotide synthesis may be performed to first yieldthe oligonucleotide with said aminoalkyl tail, to which then analkylating moiety, such as the above-noted haloacyl group or—CO—(CH₂)_(m)(X)_(n)N(R₁)—(CH₂)_(p)-L is introduced.

An exemplary preferred embodiment of an ODN-MGB conjugate which has across-linking agent attached to one of the nucleotide bases isrepresented by the formula below:

5′-GGTTATTTTTGAAGATACGAATTTCUCCAGAGACACAGCAGGATTTG TCA-CDPI₃where the underlined symbol “U” (the 26th nucleotide unit in the 50 mer)represents a 5-(3-aminopropyl)-2′-deoxyuridine which has a chlorambucilresidue attached to the amino group. The symbol “CDPI₃” represents aminor groove binder moiety as described below in connection withReaction Scheme 1. The 5-(3-aminopropyl)-2′-deoxyuridine component isincorporated into the ODN by using5′-O-trityl-5-trifluoroacetamidopropyl-2′-deoxyuridine3′-(N,N-diisopropyl-cyanoethyl-phosphoramidite in accordance with theprocedure of Gibson, K. J., & Benkovic, S. J. (1987) Nucleic Acids Res.15, 6455. The chlorambucil residue and the minor groove binder moietyare reintroduced into the ODN as described in the experimental sectionbelow.

Synthesis of Minor Groove Binder Moieties and ODN-MGB Conjugates

Presently most preferred embodiments of the minor groove binder moietiesof the present invention are “oligopeptides” derived from1,2-dihydro-3H-pyrrolo-[3,2-e)indole-7-carboxylic acid (CDPI) and from4-amino-N-methylpyrrole-2-carboxylic acid. These are synthetic peptideswhich have repeating units of the structures shown respectively inFormula 2 and Formula 4 where the degree of polymerization (m) of thepeptide is preferably 3 to 5, most preferably 5 for the peptide ofFormula 2 and 3 for the peptide of Formula 4. Reaction Scheme 1discloses a process for preparing a specific tripeptide abbreviated“CDPI₃” which thereafter can be coupled with or without minormodification, to ODNs, to form preferred embodiments of the ODN-MGBconjugates of the present invention.

Referring thus to Reaction Scheme 1, the starting material in thissynthetic scheme is3-carbamoyl-1,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylic acid or3-t-butyloxycarbonyl-1,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylicacid which can be made in accordance with the chemical literature (D. L.Boger, R. S. Coleman, and B. J. Invergo. J. Org. Chem., 1987, Vol. 52,1521-1530). The starting compounds are converted into an active ester bytreatment with the tetrafluorophenyl ester of trifluoroacetic acid(TFP-TFA). In compound 1a shown in the scheme the R group is CONH₂, in1b R is t-butyloxycarbonyl (^(t)Boc). The t-butyloxycarbonyl (^(t)Boc)group is a well known protecting group for amino functions which can beremoved by acid. The resulting activated esters 1a and 1b are reactedwith methyl 1,2-dihydro-3H-pyrroloindole-7-carboxylate (also availablein accordance with the chemical literature, see D. L. Boger, R. S.Coleman, and B. J. Invergo. J. Org. Chem., 1987, Vol. 52, 1521-1530) toyield the “dimer” peptide compounds 2a and 2e. The methyl group of thecarboxyl function is removed by treatment with base to yield the “dimer”peptides wherein the carboxylic acid group is free. This dimer isactivated once more to form an active ester with tetrafluorophenol (2ewhen R═CONH₂′, TFP-CDPI₂; and 2f when R=^(t)Boc, TFP-^(t)Boc-CDPI₂).After activation with TFP-TFA the active ester of the dimer can be usedfor forming the ODN-MGB conjugate as is described below in connectionwith the corresponding trimer. The activated ester of the dimer peptidecan also be reacted with yet another molecule of methyl1,2-dihydro-3H-pyrroloindole-7-carboxylate to form a “trimer peptide”that has its carboxylic acid function protected as a methyl ester, 3a(methyl 3-carbamoyl-11,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylatetrimer). The methyl group is removed by treatment with base and theresulting “trimer peptide” 3b is converted again into an activetetrafluorophenyl ester 3c (2,3,5,6-tetrafluorophenyl3-carbamoyl-1,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylate trimer,TFP-CDPI₃). The active tetrafluorophenyl ester 3c can be used to furtherlengthen the peptide chain by repeating the steps of reacting withmethyl 1,2-dihydro-3H-pyrroloindole-7-carboxylate, saponifying theresulting methyl ester, and if desired, reacting with TFP-TFA again tomake the active tetrafluorophenyl ester of the peptide incorporating4-CDPI moeieties. As it will be readily understood, these steps can berepeated further until the desired number of CDPI Moieties are includedin the peptide. In the herein described preferred embodiments the activetetrafluorophenyl ester of the tripeptide 3c (TFP-CDPI₃) is utilized forcoupling to an ODN to make an ODN-MGB, or for synthesizing an ODN-MGB ona suitable modified controlled pore glass (CPG) solid support as isdescribed below in connection with Reaction Schemes 4 and 5. ReactionScheme 1 indicates as its last step the preparation of ahydroxylpropylamide derivative from the active tetrafluorophenyl esterof the tripeptide 3c (TFP-CDPI₃). The hydroxylpropylamide derivative ofthe tripeptide 3d(3-carbamoyl-1,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carbox]-1-amido-3-propanoltrimer, CDPI₃-3-hydroxylpropylamide) can be used for coupling with anODN to obtain an ODN-MGB in accordance with the present invention. Thetripeptide 3d however, was also used as a “free standing” minor groovebinder molecule as a control in certain binding studies which aredescribed below.

Referring now to Reaction Scheme 2 the synthesis of another preferredembodiment of the minor groove binder peptides is disclosed, where the“monomer” is the residue of 4-amino-N-methylpyrrol-2-carboxylic acid,and which embodiment also bears a reporter group/containing adiazobenzene moiety. Thus, in accordance with this scheme6-[(tert-butyloxy)carboxamido]hexanoic acid is condensed in the presenceof N,N-dicyclohexylcarbodiimide with2-[4-(phenylazo)-benzylthio]-ethanol to form(2-[4-(phenylazo)benzylthio]ethyl 5-(tert-butyloxy)carboxamido]pentylcarboxylate, 11). The ^(t)Boc protecting group isremoved from compound 11 by treatment with trifluoroacetic acid (TFA)and the resulting compound having a free amino function is reacted withan activated ester of ^(t)Boc protected4-amino-N-methylpyrrol-2-carboxylic acid. The latter activated estercompound (1,2,3-benzotriazol-1-yl1-methyl-4-(tert-butyloxy)carboxamido-pyrrole-2-carboxylate) is madefrom 1-methyl-4-[tertbutyloxy)carboxamido]pyrrole-2-carboxylic acidwhich is available pursuant to the literature procedure of L. Grehn, V.Ragnarsson, J. Org. Chem., 1981, 46, 3492-3497. The resulting2-[4-(phenylazo)benzylthio]ethyl5-[1-mdthyl-4-(tert-butyloxy)carboxamido]pyrrole-2-carboxamido]pentylcarboxylate,12) has one unit of the monomer “2-amino-N-methylpyrrol carboxylic acid”residue attached to the reporter group that carries the diazobenzenemoiety. After removal of the ^(t)Boc protecting group withtrifluoroacetic acid and coupling with one or more molecules of1,2,3-benzotriazol-1-yl1-methyl-4-(tert-butyloxy)carboxamido-pyrrole-2-carboxylate can beaccomplished, until a peptide containing the desired number of monomerresidues is obtained. Such a compound having n number of monomers and afree amino group is indicated in Reaction Scheme 2 as 16a. Compound 16acan be reacted with an activated ester (such as a1,2,3-benzotriazol-1-yl activated ester) of ^(t)Boc protected6-aminohexanoic acid to provide the oligopeptide shown as compound 16bin Reaction Scheme 2. The ^(t)Boc protecting group can be removed fromthe latter compound under acidic conditions, and the resultingderivative having a free amino function can be attached by conventionalsynthetic methods to either the 3′-phosphate or 5′-phosphate end of anODN. Alternatively, the derivative having a free amino function can alsobe attached to the 3′ or 5′-OH end of an oligonucleotide using a varietyof bifunctional linking groups, as discussed above.

Referring now to Reaction Scheme 3a general method for coupling a3′-amino tailed or 5′-aminotailed ODN with the tetrafluorophenyl (TFP)ester activated exemplary minor groove binding oligopeptides isillustrated. Although the scheme shows the use of the TFP activatedexemplary minor groove binding compounds obtained in accordance withReaction Scheme 1, it should be kept in mind that this general method issuitable for the coupling of other TFP activated minor groove bindingcompounds with ODNs, as well. The reference numeral 1a through 3c inReaction Scheme 3 refer to the exemplary compounds obtained inaccordance with Reaction Scheme 1.

The 3′- or 5′-amino tailed ODNs can be synthesized by conventionalmethods; for example an aminohexyl residue can be attached to either endof the ODN by using commercially available N-monomethoxytritylaminohexylphosphoramidite. Alternatively, the amino tailed ODNs can be synthesizedin accordance with the methods described in U.S. Pat. No. 5,419,966, thedisclosure of which is expressly incorporated herein by reference. Inaccordance with the present scheme the amino tailed ODN is convertedinto a cetyltrimethylammonium salt to render it soluble in organicsolvents, and the tetrafluorophenyl ester activated minor groove bindermolecule is condensed therewith, preferably in DMSO as a solvent.

Reaction Scheme 4 discloses another method of coupling an active esterof a minor groove binder molecule to a 5′-amino tailed ODN. The exampleshown in the scheme is that of the TFP ester of the tripeptide derivedfrom 3-carbomoyl-1,2-dihydro-3Hpyrrolo[3,2-e]indole-7-carboxylic acidresidues (TFP-CDPI₃) but it should be understood that the genericprinciples disclosed in connection with this reaction scheme can be usedwith other minor groove binder molecules as well. In this method, theODNs still attached to a CPG support, and has a free amino group on its“amino tail”. This can be obtained by usingN-monomethoxytritylazinohexyl phosphoramidite mentioned above. Themonomethoxytrityl group is removed after the coupling of thephosphoramidite to give the desired CPG-bearing-“amino-tailed ODN”.Alternatively, such a CPG can be obtained in accordance with thedisclosure of U.S. Pat. No. 5,419,966, and references cited therein. Byway of summary, the ODN is synthesized stepwise attached to the CPG, andhaving a tail having an amino group protected with a9-fluorenylmethoxycarbonyl (Fmoc) group. After the desired sequence ofnucleotides has been built up, the Fmoc group is removed from the aminogroup while the ODN is still attached to the CPG support. In accordancewith Reaction Scheme 4 of the present invention this“CPG-bearing-amino-tailed-ODN” having the free amino group is condensedwith the active ester (TFP-CDPI₃, 3c) or with a like activated form of aminor groove binder. The ODB-MGB conjugate is thereafter removed fromthe CPG support by conventional methods, most frequently by treatmentwith ammonia.

Reaction Scheme 5 discloses another preferred method for preparing, theODN-MGBs of the present invention. More particularly, Reaction Scheme 5discloses the preferred synthetic process for preparing ODN-MGBs byfirst attaching a linking molecule to a CPG support, thereafterattaching an activated form of minor groove binder to the linkingmolecule, and thereafter building the ODN of desired sequencestep-by-step in an automatic ODN synthesizer using the just describedmodified CPG support. The ODN-MGB conjugate is removed from the CPGsupport only after the ODN moiety of desired sequence has beencompleted. The linking molecule in this case is a trifunctionalmolecule, with each function having different reactivity, which permitattachment to the CPG, reaction with the activated form of minor groovebinder moiety and the building of the ODN portion, each using adifferent functionality of the linking molecule. A more general anddetailed description of this synthetic method and of the trifunctionallinking molecules which can be utilized in the method, but without anyreference to minor groove binders, can be found in U.S. Pat. No.5,419,966, the disclosure of which is expressly incorporated herein byreference. Reaction Scheme 5 illustrates this synthetic process with theexample of β-alanilyl-3-amino-1,2-propanediol as the trifunctionallinking molecule, and TFP-CDPI₃ (compound 3c) as the activated form ofthe minor groove binder.

Thus in accordance with Reaction Scheme 5, Fmoc protected β-alanine isreacted with tetrafluophenyl trifluoroacetate (TFP-TFA) to provide2,3,5,6-tetrafluorophenyl3-[N-(9-fluorenylmethoxycarbonyl)]aminopropionate (4). The active ester4 is reacted with 3-amino-1,2-propanediol to provide1-[3-[N-(9-fluorenylmethoxycarbonyl)amino]-1-oxopropyl]amino-(R,S)-2,3-propanediol(5). The primary hydroxyl group of 5 is thereafter protected with adimethoxytrityl group to give1-[3-[N-(9-fluorenylmethoxycarbonyl)amino]-1-oxopropyl]amino-(R,S)-2[[bis(methoxyphenyl)phenylmethoxy]methyl]-2-ethanol(6). The secondary hydroxyl group of compound 6 is reacted with succinicanhydride and the carboxylic group in the resulting compound isthereafter converted into an active ester, 2,3,5,6-tetrafluorophenyl1-[3-[N-(9fluorenylmethoxycarbonyl)amino]-1-oxopropyl]amino(R,S)-2-[[bis(methoxyphenyl)phenylmethoxy)methyl]-2-ethylbutanedioate (7). Compound 7 is then attached to a long chain aminoalkylcontrolled pore glass support (LCAA-CPG, or alkylamine CPG) which iscommercially available and is described in the above-cited U.S. Pat. No.5,419,966. The resulting “modified CPGII is shown in Reaction Scheme 5as Compound 8. The Fmoc protecting group is removed from 8 by treatmentwith mild base (piperidine in dimethylformamide) to yield the “modifiedCPG” 9 that has a free primary amine function as part of the linkingmolecule. In the next step the activated minor groove binder molecule,in this instance TFP-CDPI₃ (compound 3c) is reacted with the primaryamine function of 9, to yield the modified CPG 10 that includes theminor groove binder moiety and still has the primary hydroxyl group ofthe linking group protected with a dimethoxytrityl group. Although thisis not shown in Reaction Scheme 5, in the subsequent steps thedimethoxytrityl group is removed and the ODN synthesis is performed inan automatic synthesizer, by steps which are now considered conventionalin the art.

When the synthesis is complete the ODN-MGB conjugate is removed from theCPG support by treatment with ammonia. The latter step cleaves the bondattaching the secondary hydroxyl group of the 3-amino-1,2-propanediolmoiety to the CPG support.

Biological Testing and Discussion

The ODN-MGB conjugates bind to single stranded DNA. They also bind todouble stranded DNA in the presence of a recombinase enzyme, and in somecases to single stranded RNA and DNA and RNA hybrids as well. Thebinding however occurs only if the ODN moiety is complementary, orsubstantially complementary in the Watson-Crick sense, to a targetsequence in the target DNA or RNA. When this condition is met, thebinding of the ODN-MGB to the target sequence is significantly strongerthan binding of the same ODN would be without the minor groove binder.The foregoing is demonstrated by the tests described below, and providesutility for the ODN-MGB conjugates of the present invention asanalytical and diagnostic hybridization probes for target DNA or RNAsequences, and in therapeutic antisense and anti-gene applications.

TABLE 1 T_(m)'s data of the (dAp)₈ + (dTp)₈ duplex carryingintercalators or oligo-(1-methyl-2-carboxy-4amino)pyrrole residuesattchcd to 3′-end of the ODN.^(a) COMPLEX T_(m) ΔT_(m) ^(b) (dAp)₈ +(dTp)₈ 21.1 — (dAp)₈ + (dTp)₈ + Distamycin A^(c) 47.1 26.0 (dAp)₈ +(dTp)₈-X_(m) m = 2 39.4 18.3 m = 3 51.7 30.6 m = 4 60.2 39.1 m = 5 65.444.3 (dTp)₈ + (dAp)₈-X_(m) m = 2 29.1 8.0 m = 3 39.0 17.9 m = 4 42.721.6 m = 5 52.6 31.5 (dAp)₈-Y + (dTp)_(s) 30.5 9.4 (dAp)₈-Y +(dTp)₈-Y^(d) 42.9 21.8 ^(a)Reported parameters are averages of at leastthree experiments. Optical melts were conducted in 0.2 M NaCl, 0.3 mMEDTA, 0.01 M (±0.1° C.) Na₂HPO_(4′) pH 7.0 with [(dTp)₈•(dAp)₈ ] =2.5•10⁻⁵ M. ^(b)The difference in T_(m) between modified and unmodifiedduplexes. ^(c)Concentration of distamycin A was 2.5•10⁻⁵ M. ^(d)Ethidiumbromide (EtBr) was conjugated by its 8-NH₂-position to the 3′-terminalphosphate of the ODNs through a β-alanine linker by the method in ref12.

Table 1 illustrates the melting temperature of several complexes formedof complementary oligonucleotides which have the minor groove bindermoiety derived from 4-amino-N-methylpyrrol-2-carboxylic acid residues.The minor groove binder moiety is specifically shown as the radical X bythe formula below Table 1. It is noted that the radical X also includesa linking moiety which is derived from 6-aminohexanoic acid. Theoligonucleotides utilized here are 8-mers of 21deoxyadenylic acid, and8-mers of thymidylic acid. The minor groove binder X is attached to theODNs at the 3′-phosphate end, the 5′-end of these ODNs have nophosphate. In this regard it is noted that the ODNs are abbreviated inthese and the other tables in the manner customary in the art. The groupY symbolizes an ethidium bromide moiety attached to the 3′phosphate endthrough a “β-alanine” linking moiety. The Y group represents anintercalating group and acts as a control for comparison with the minorgroove binding groups. The symbol a represents the number of4-amino-N-methylpyrrol-2-carboxylic acid residues present in eachODN-MGB of the table.

As is known in the art, the melting temperature (T_(m)) of anoligonucleotide or polynucleotide duplex is defined as that temperatureat which 50% of the respective oligonucleotide or polynucleotide isdissociated from its duplex, Watson Crick hydrogen bonded form. A highermelting temperature (T_(m)) means a more stable duplex. As is knownfurther, the melting temperature of an oligonucleotide or polynucleotideis dependent on the concentration of the nucleotide in the solution inwhich the melting temperature is measured, with higher concentrationsresulting in higher measured melting temperatures. The meltingtemperatures indicated in these tables were measured under conditionsindicated in the table and in the experimental section. ΔT_(m)represents the change in melting temperature of the modified duplexrelative to the melting temperature of the (dAp)_(g).(dTp)₈ complexwhich has no minor groove binder moiety.

As it can be seen from Table 1, the covalently bound minor groove bindermoiety significantly increases the stability (melting temperature T_(m))of the complex, whether the group X (minor groove binder moiety) isattached to the (dTp)₈ or to the (dAp)₈ oligonucleotide. In thisinstance the greatest degree of stabilization (highest meltingtemperature) is achieved when the minor groove binder moiety is a 5-meroligopeptide. In the comparative experiment when the intercalating groupY is attached to the (dAp)_(g) oligomer, a comparatively much smallerdegree of stabilization is attained. Even attaching the intercalating Ygroup to each of the two strands of oligomers in this experiment, raisedthe melting temperature less than the minor groove binder moiety havingfive 4-amino-N-methylpyrrol-2-carboxylic acid residues.

TABLE 2 T_(m)'s data of the duplexes formed by hexadeca-,octathymidylate and their oligo-(1-methyl-2-carboxy-4amino)pyrrolederivatives with polydeoxyriboadenylic acid in 0.2 M NaCl, 0.01 Mna₂HPO₄, 0.1 mM EDTA (pH7.0). X is same as Table 1. Oligo DerivativeT_(m)° C. ΔT_(m)° C. (dTp)₁₆ 48.5 — (dTp)₁₆-NH(CH₂)₆COOH 49 0.5(dTp)₁₆-X m = 1 49.3 0.8 m = 2 55.6 7.1 m = 3 61 12.5 m = 4 66 17.5 m =5 68 19.5 (dTp)₈ 28 — (dTp)₈-X m = 1 28 0 m = 2 40 12 m = 3 52 24 m = 460 32 m = 5 66 38

Table 2 discloses information in a manner similar to Table 1. In thetests reported in this table 16-mer ODNs of thymidylic acid having theminor groove binder moiety represented by X (X is the same as inTable 1) were complexed with polydeoxyriboadenylic acid. As acomparative control a 16 mer ODN of thymidylic acid (dTp)₁₆ connected atits 3′-phosphate end to 6-aminohexanoic acid was also tested.Additionally an 8-mer of thymidylic acid (dTp)₈ and its conjugates withthe minor groove binders of varying peptide length were also tested. Inthese tests too, the minor groove binder attached to the ODN causessignificant stabilization of the complex between the ODN-MGB and thecomplementary DNA strand. Greatest stabilization occurs when the numberof 4-amino-N-methylpyrrol-2-carboxylic acid residues in the minor groovebinder moiety is five. In contrast, the aminohexanoic acid tail on the16-mer ODN results in virtually no stabilization of the complex.

TABLE 3 Melting temperatures (° C.) of duplexes formed by poly(dA) andpoly(rA) with (Tp)₈ strands terminallylinked to CDPI₁₋₃ and BocDPI₁₋₂ligands.^(a) Octathymidylate poly(dA) poly(rA) derivative T_(m) ΔT_(m)T_(m) ΔT_(m) (dTp)₇dTp-L1 25 — 13 — (dTp)₇dTp-L1-X m − 1 34  9 18 5(dTp)₇dTp-L1-X m = 2 50 25 —^(b) — (dTp)₇dTp-L1-X m = 3 68(65) 43(40)32(31) 19(18) (dTp)₇dTp-L1-Y m = 1 26  1 12 −1   (dTp)₇dTp-L1-Y m = 2 4318 17 4 L1-pdT(pdT)₇ 24 — 12 — X-L1-pdT(pdT)₇ m = 1 31  7 14 2X-L1-pdT(pdT)₇ m = 2 49 25 —^(b) — X-L1-pdT(pdT)₇ m = 3 68 44 35 23 Y-L1-pdT(pdT)₇ m = 1 23 −1 9 −3   Y-L1-pdT(pdT)₇ m = 2 41 17 19 7^(a)The data in brackets were obtained for the derivative with linkderL2. ^(b)No melting transition was observed.

Table 3 discloses melting temperature (T_(m)) and change in meltingtemperature (ΔT_(m)) data in tests where the oligonucleotide is an 8-merof thymidylic acid having a minor groove binder moiety attached to iteither at the 5′-phosphate or 3′-phosphate end, as indicated in thetable. The minor groove binder moieties represented here by X and Y are“oligopeptides” based on the residue of1,2-dihydro-3Hpyrrolo[3,2-e]indole-7-carboxylic acid (CDPI or BocDPI)and their structures are shown in the table. These minor groove bindingoligopeptides are attached to the ODN through a linking moiety “L1 orL2” the structures of which are also shown below the table. The ODN-MGBconjugates were incubated with a complementary ribo- or deoxyribohomopolymer. Thus for ODN-MGB conjugates comprising ODNs of thymidylicacid, poly A or poly-dA was used. The change in melting temperature(ΔT_(m)) is indicated relative to the complex with the ODN which bearsthe corresponding linking group L1 or L2 in the corresponding end of theODN, but bears no minor groove binding moiety. As it can be seen fromTable 3, these ODN-MGB complexes again exhibit significant stabilizationof the complex with the complementary deoxyribo homopolymer, with thegreatest stabilization occurring in these tests when the minor groovebinding moiety has 3 CDPI units. Surprisingly, stabilization of thecomplex occurs even when the ODN-MGB is incubated with a complementaryribohomopolymer. This is surprising because it had been generallyobserved in the prior art that free standing minor groove bindingmolecules do not bind to DNA-RNA hybrids.

TABLE 4 T_(m)'s data (° C.) of heterogeneous duplexes phosphodiester andphosphorothioate backbones and oligo(pyrroloindole carboxainide) peptideresidues attached to the different positions^(a.) Derivative Derivativeof ApGpCpGpGpApTpG of CpApTpCpCpGpCpT DNA Type of Type of terminal3′-L2—X & 2′-DNA PS^(c) Backbone modification 3′-L1— 3′-L2—X 5′-X—L1—5′-X—L1— none^(b) 5′-X—L1— 3′-L2—X DNA 3′-LI— 41 52 45 50 33 27 403′-L2—X 57 81 78 77 50 73 77 5′-X—LI— 58 79 76 76 49 70 75 3′-L2—X5′-XL1— 60 72 — 65 — — — 2′-DNA PS^(c) none^(b) 32 43 32 — 24 16 285′-X—L1 38 69 67 — 28 62 63 3′-L2—X 45 74 71 — 36 64 69^(a)Concentration of ODNs in the melting mixtures was 2 × 10⁻⁶ M in 140mM KCl, 10 mM MgCl₂, 20 mM HEPES-HCl (pH 7.2). ^(b)The ODN has free 3′-and 5′-OH groups. ^(c)PS is phosphorothioate linkage.

L1 = —O(CH₂)₆NH— L2 = —OCH₂CH(OH)CH₂NHCOCH₂CH₂NH—

Table 4 discloses results of a study of duplex formation betweenderivatives of two complementary octamers: CpApTpCpCpGpCpT andApGpCpGpGpApTpG. Each octamer was modified, as shown in the table, sothat hybridization of the corresponding oligodeoxyribonucleotides and ofoligodeoxyribonucleotides having a phosphorothioate backbone wereexamined. The ODN also had the tripeptide based on the residues of1,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylic acid (CDPI) (minorgroove binder moiety X) attached either to the 3′ or to the 5′ end (asindicated) through a linking group of the structure L1 or L2. (X, L1 andL2 are the same as in Table 3.) As controls, the melting temperature ofduplexes was also determined for duplexes where the ODNs bore only thelinking groups. As it can be seen in the table, the duplexes aresignificantly stabilized by the presence of a minor groove bindermoiety, and greater stabilization occurs when each strand of the duplexhas a covalently bound minor groove binder.

TABLE 5 T_(m)'s data (° C.) of heterogeneous duplexes carrying3′-oligo(pyrroloindole carboxamide) peptide residues. Complemenaryd(AGCGGATG)p d(AICILATI)p ODNs 3′-L1— 3′-L2—X 3′-L1— 3-L1—X d(CATCCGCT)p3′-L1— 41 52 11 — 3′L2—X 57 81 48 67 d(CATCCICT)p 3′-L1— 31 48  0 413′L1—X 54 79 48 63

L1 = —O(CH₂)₆NH— L2 = —OCH₂CH(OH)CH₂NHCOCH₂CH₂NH—

Table 5 discloses melting temperature data obtained when complementaryor “quasi complementary” ODN-MGB were incubated and examined for duplexformation. The minor groove binding moiety X and the linking groups L1and L2 are shown in the table and are the same as in Tables 3 and 4. Asanticipated, when guanine is replaced inosine (I) in the strands thebinding of the duplexes is very weak (T_(m) is approximately 0° C.) ifthere is no minor groove binding moiety present. However, when guanineis replaced by inosine in the oligonucleotides the presence of onecovalently appended minor groove binder X stabilized the hybrid byalmost 50° C. and the presence of two such minor groove binders inantiparallel orientation provided 63° C. of stabilization. When the samestrands contained guanine, one minor groove binder increased, —) theT_(m) by 15° C. while two increased it by nearly 45° C. To the knowledgeof the present inventors a T_(m) of 81° C. for an 8 mer is unprecedentedin the prior art.

Primer Extension Experiment.

That sequence specificity in the Watson-Crick sense of the ODN portionof the ODN-MGB conjugate is required for complexing the ODN-MGBconjugate to a target sequence was demonstrated by a primer extensionexperiment. In this experiment, primer extension occurs with the enzymeT7 DNA polymerase that works from the 5′ end of a template strand. A16-mer ODN-MGB which was complementary in the Watson Crick sense to atarget sequence on the template strand was incubated with a long singlestranded DNA template and the T7 DNA polymerase enzyme. Blockage of theprimer extension was noted at the site of binding with the ODN-MGB whenthe minor groove binding moiety was on the 5′ end of the 16-mer ODN. Theminor groove binder was the pyrroloindole tripeptide shown in thisapplication in Table 5. When there was a single mismatch in the sequencespecificity of the 16-mer to the target, primer extension was notblocked. Primer extension was also not blocked when the minor groovebinder moiety was attached to the 3′ end of the 16-mer. Primer extensionwas also not blocked when the sequence specific 16-mer and the freeminor groove binder molecule (Compound 3d, not covalently attached tothe ODN) was incubated with the template and the enzyme. Theseexperiments show that sequence specificity of the ODN-MGB is importantfor complex formation, and that the minor groove binding moiety does notsimply act as an “anchor” to non-specifically bind the appended ODN toanother strand. The ability of ODN-MGB conjugates to inhibit primerextension indicates that these conjugates can be used diagnostically aspolymerase chain reaction (PCR) clamping agents. (See Nucleic AcidResearch (1993) 21: 5332-5336).

Slot-Blot Hybridization Assay

The ODN-MGB conjugates of the present invention are useful ashybridization probes. This is demonstrated by the description of thefollowing experiment utilizing a ³²P-labeled ODN-MGB conjugate as adiagnostic probe. When compared to the same ODN without a covalentlylinked minor groove binder (MGB) moiety, the conjugate hybridizes to itscomplement with greater strength, efficiency and specificity. Theslot-blot hybridization assay is a widely used DNA probe diagnosticassay, and the attributes of these MGB-ODN conjugates improve theperformance of the assay.

Specifically, in the herein described experiment a standard protocol wasfollowed, as described in Protocols for Nucleic Acid Blotting andHybridization, 1988, Amersham, United Kingdom. Labelled test ODN whichhybridized to the immobilized plasmid was quantitated as counts perminute (cpm), and plotted vs temperature of hybridization. Four 16-merprobes complementary to M13mp19 DNA (a phage DNA) were evaluated. Two ofthese probes were totally complementary to a site in the phage DNA; oneof these contained a 3′-conjugated CDPI₃ moiety while the other wasunmodified. The second pair of probes were targeted to the same site inM13mp19 DNA but each contained a single mismatch (underlined in drawingFIG. 1). Here again, one ODN was 3′-conjugated to CDPI₃ while the otherwas unmodified.

The results of the slot hybridization study are shown in FIG. 1.Compared to an unmodified but otherwise identical 16-mer, theCDPI₃-containing probe formed a hybrid with a melting temperature(T_(m)) of 50° C. versus only 33° C. This higher melting temperaturemore than doubled the yield of perfectly matched hybrids. When amismatch was introduced into either probe, stability of the respectivehybrids dropped. The CDPI₃-modified probes exhibited good sequencediscrimination between 37°-50° C. Furthermore, under the hybridizationconditions used here there was no evidence for binding of the CDPI₃moiety to preexisting double-stranded regions in the M13mp19. DNAtarget, indicating that totally non-specific binding of these conjugatesis not present.

Sequence-Specific Alkylation of a Gene in Cultured Human Cells

The ODN-MGB conjugates of the present invention which also bear acovalently attached alkylating agent can be used as “anti-gene” agents,that is for the suppression of the expression of undesired (diseasecausing) genes, provided the ODN-MGB conjugate is complementary to atarget sequence in the target gene. In such a case the NGB moietyimproves the binding to the double stranded gene (in the presence of arecombinase enzyme) and the alkylating moiety results in permanentcovalent binding of the ODN-MGB conjugate to the target sequence.

As a demonstrative experiment the above described 50-mer ODN which was3′ end-modified with a CDPI₃ group and internally modified with anitrogen mustard group (chlorambucil) sequence-specifically crosslinkedto the expected site in a targeted gene (HIA DQβ1 0302 allele) presentin living human BSM cells (a human B-lymphocyte cell line). The ODN-MGBconjugate was added to a suspension of BSM cells at 1-50 μM finalconcentration. After 3.5 hr the genomic DNA was extracted and treatedwith hot pyrrolidine to convert any alkylation events into nicks. Nextthe targeted region of the 0302 allele was amplified by LM-PCR (ligationmediated-polymerase chain reaction), a technique which can be used todetect cleavage events at single-base resolution. Analysis of theproducts on a sequencing gel showed that the modified ODN had bound toand alkylated the targeted site. A similar ODN lacking the CDPI₃ groupwas considerably less effective in efficiency of alkylation of thetarget.

It is probable that in the experiment above the recognition and bindingof the ODN-MGB conjugate to homologous double-stranded DNA took placewith the assistance of nuclear recombinases. In like experiments andapplications endogenous recombinase enzymes can catalyze the sequencespecific targeting of double-stranded genomic DNA by ODN-CDPI₃conjugates in other living cells. When these ODNs have an appendedcrosslinking agent, they can alkylate the targeted DNA. By stabilizingthe D-loop formed in the presence of recombinase, the CDPI₃ promotes thecrosslinkage reaction. The crosslinkage event is a powerful inhibitor ofexpression of the targeted gene. Thus crosslinking ODN-CDPI₃ conjugatescan be used as antigene agents.

SPECIFIC EMBODIMENTS Experimental Section

General Experimental

All air and water sensitive reactions were carried out under a slightpositive pressure of argon. Anhydrous solvents were obtained fromAldrich (Milwaukee, Wis.). Flash chromatography was performed on 230-400mesh silica gel. Melting points were determined on a MeI-Temp meltingpoint, apparatus in open capillary and are uncorrected. Elementalanalysis was performed by Quantitative Technologies Inc. (Boundbrook,N.J.). UV-visible absorption spectra were recorded in the 200400-nmrange on a UV-2100 (Shimadzu) or a Lambda 2 (Perkin Elmer)spectrophotometers. ¹H NMR spectra were run at 20° C. on a Bruker WP-200or on a Varian XL-200 spectrophotometer; chemical shifts are reported inppm downfield from Me₄Si.

2,3,5,6-Tetrafluorophenyl3-carbamoyl-1,2-dihydro-3H-pyrrolor[3.2-e]lindole-7-carboxylate (1a).2,3,5,6-Tetrafluorophenyl trifluoroacetate (2.6 g, 10 mmol, H. B.Gamper, M. W. Reed, T. Cox, J. S. Virosco, A. D. Adams, A. A. Gall, J.K. Scholler and R. B. Meyer, Jr. Nucleic Acids Res., 1993, Vol. 21, No.1, 145-150) was added dropwise to a solution of3-carbamoyl-1,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylic acid (1.4g, 6.1 mmol, D. L. Boger, R. S. Coleman, and B. J. Invergo. J. Org,Chem., 1987, Vol. 52, 1521-1530) and triethylamine (1.4 ml, 10 mmol) in15 ml of anhydrous DMF. After 1 hr, the reaction mixture wasconcentrated under vacuum (0.2 mm). The residue was triturated with 2 mlof dry dichloromethane. Ethyl ether (50 ml) was added and the mixturewas left at 0° C. overnight. The precipitate was collected by filtrationon sintered glass funnel, washed first with 50% ether/CH₂cl₂ (10 ml),then with ether (50 ml) and dried in vacuo. The product was obtained asa yellow solid (1.8 gi 75%): ¹H NMR (Me₂SO-d ₆, 200 MHz, ppm) 12.32 (s,1H, NH), 8.13 (d, 1H, J=9 Hz, C4-H), 8.01 (m, 1H, C₆F₄H), 7.41 (s, 1H,C8-H), 7.26 (d, 1H, J=9 Hz, C5-H), 6.17 (s, 2H, CONH₂), 3.99 (t, 2H, J=9Hz, NCH₂CH₂), 3.30 (t, 2H, J=9 Hz, NCH₂CH₂). Anal. Calcd. forC₁₈H₁₁N₃0₃F₄×2H₂0: C, 50.3; H, 3.52; N, 9.7. Found; C, 50.81; H, 3.60;N, 9.95. 2,3,5,6-Tetrafluorphenyl3-(tert-butyloxycarbonyl)-1,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylate(1b). 2,3,5,6-Tetrafluorophenyl trifluoroacetate (2.6 g, 10 mmol) wasadded dropwise to a solution of3-(tertbutyloxycarbonyl)-1,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylicacid (1.0 gi 3.7 mmol, D. L. Boger, R. S. Coleman, and B. J. Invergo. J.Org. Chem., 1987, Vol. 52, 1521-1530) and triethylamine (1.5 ml, 10mmol) in 10 ml of anhydrous CH₂cl₂. After 4 hrs, CH₂cl₂ was removed byevaporation at reduced pressure. Flash chromatography (4×2O cm,hexane-ethyl acetate, 1:2) afforded 1b as a yellow crystalline solid(1.25 gr 75%): ¹H NMR (Me₂SO-d ₆, 200 MHz, ppm) 12.39 (d, 1H, J=1.4 Hz,NH), 8.02 (m, 1H, C₆F₄H), 7.9 (br s, 1H, C₄H)i, 7.45 (d, 1Hj, J=1.4 Hz,C8-H), 7.33 (d, 1H, J=9 Hz, C5-H), 4.02 (t, 2H, J=9 Hz, NCH₂CH₂), 3.26(t, 2H, J=9 Hz, NCH₂CH₂ 2)″ 1.51 (s, 9H, C(CH₃)₃). Anal. Calcd. forC₂₂H₁₈N₂0₄F₄: C, 58.67; HI, 4.03; N, 6.22. Found: C, 58.45; HI, 4eO9; N,6*13a

3-carbamoyl1,2-dihydro-3H-pyrrolor3,2-elindole-7 carboxylate dimermethyl ester (2a). A solution of methyl1,2-dihydro-3H-pyrroloindole-7-carboxylate (0.6 g, 1.5 mmol), 1a (0.45g, 2.25 mmol) and triethylamine (0.2 ml, 1.4 mmol) in 10 ml of anhydrousDMF was incubated at RT for 24 hrs and then at 0° C. for 12 hrs. Theresulting insoluble solid was collected by filtration, washed with DMF(10 ml) and ether (20 ml). Drying in vacuo afforded 2a (0.61 g, 91%) asa pale yellow solid: (¹H NMR (Me₂SO-d ₆, 200 MHz, ppm) 12.00 (d, 1H,J=1.8 Hz, NH′), 11.54 (s, 1H, NH), 8.28 (d, 1H, J=9 Hz, C4′-H), 7.97 (d,1H, J=9 Hz, C4-H), 7.33 (d, 1H, J=9 z, C5′-H), 7.22 (d, 1H, J=9 z,C5-H), 7.13 (d, 1H, J=1.4 Hz, C8′-H), 6.94 (d, 1H, J=1.1 Hz, C8-H), 6.01(s, 2H, CONH₂), 4.62 (t, 2H, J=8 Hz, (NH₂CH₂)′), 3.98 (t, 2H, J=8 Hz,NCH₂CH₂), 3.88 (s, 3H, CH₃), 3.41 (t, 2H, J=8 Hz, (NCH₂CH₂)1), 3.29 (t,2H, NCH₂CH₂, partially obscured by water). Anal. Calcd. forC₂₄H₂₁N₅O₅×1H₂O×1DMF: C, 58.69; HI, 5.84; N, 15.21. Found: C, 58.93; H,5.76; N, 15.82.

3-(tert-Butyloxycarbonyl)-1,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylatedimer methyl ester (2c). A solution of methyl1,2-dihydro-3H-pyrroloindole-7carboxylate (0.5 g, 2.5 mmol), 1b (1.0 g,2.2 mmol) and triethylamine (0.1 ml, 0.7 mmol) in 10 ml of anhydrous DMFwas incubated at RT for 10 hrs and at 0° for 12 hrs. The resultinginsoluble solid was collected by filtration, washed with DMF (5 ml) andether (40 ml). Drying in vacuo afforded 2c (0.81 g, 74%) as an off whitesolid: ¹H NMR (Me₂SO-d ₆, 200 MHz, ppm) 12.01 (s, 1H, NH′), 11.64 (s,1H, NH), 8.28 (d, 1H, J=9 Hz, C4′-H), 7.8 (br s, 1H, C4-H), 7.32(apparent t, 2H, C5′-H+C5-H), 7.13 (d, 1H, J=1.1 Hz, C8′-H), 6.98 (d,1H, J=1.1 Hz, C8-H), 4.62 (t, 2H, J=S Hz, (NH₂CH₂)′),′ 4.02 (t, 2H, J=SHz, NCH₂CH₂), 3.88 (s, 3H, CH₃), 3.41 (t, 0.2H, - Hz, (NCH₂CH₂)1), 3.25(t, 2H, NCH₂CH₂), 1.52 (so 9H, C(CH₃)). Anal. Calcd. for C₂₉H₂₉N₄0₅: C,67.19; H, 5.64; N, 11.19. Found: 66.72, H, 5.69; N, 11.31.

2,3,5,6-Tetrafluorophhenyl 3-carbamoyl-1,2-dihyo-Ryrrolor3o2-elindole-7-carboxylate dimer (2e).2,3,5,6-Tetrafluorophenyl trifluoroacetate (2.6 g, 10 mmol) was addeddropwise to a suspension of 2b (1.2 g, 2.8 mmol, D. L. Boger, R. S.Coleman, and B. J. Invergo. J. Org. Chem., 1987, Vol. 52, 1521-1530) in15 ml of anhydrous DMF. Triethylamine (1.4 ml, 10 mmol) was added andthe mixture was stirred for 3 hrs. The mixture was concentrated in vacuo(0.2 mm) using rotary evaporator. The residue was triturated with 20 mlof dry dichloromethane. The product obtained was filtered, washed withdichloromethane (10 ml), ether (20 ml), and dried in vacuo to give 2e asa yellow solid (1.5 g, 93%): (¹H NMR (Ne₂SO-d ₆, 200 MHz, ppm) 12.51 (do1H, J=1.8 Hz, NH′), 11.58 (s, 1H. NH), 8.39 (d, 1H, J=8.9 Hz, C4′H),8.04 (m, 1H, C₆F₄H)t 7.98 (do 1H, J=S.S Hz, C4-H), 7.58 (s, 1H, C8′),7.42 (d, 1h, J=9 Hz, C5′-H), 7.22 (d, 1H, J=9 Hz, C5-H), 6.98 (s, 1H,CB—H), 6.11 (s, 2H, CONH₂), 4.66 (t, 2H, J=7.8 Hz, (NCH₂CH₂)1), 3.94 (t,2H, J=9.1 Hz, NCH₂CH₂), 3.47 (t, 2H, J=8 Hz, (NCH₂CH₂)′), 3.29 (t, 2H,J=9.1 Hz, NCH₂CH₂). Anal. Cacld. for C₂₉H₁₉N₅O₄F₄×1.5H2O: C, 57.62; H,3.67; N, 11.59. Found: C, 57.18; H, 3.31; N, 11.54.

2,3,5,6-Tetrafluoroiphenyl3-(tert-butyloxycarbonyllls2-dihydro-3H-Ryrrolor3o2-elindole-7-carboxylatedimer (2f). 2,3,5,6-Tetrafluorophenyl trifluoroacetate (0.75 g, 2.9mmol) was added dropwise to a suspension of 2d (0.25 g, 0.5 mmol, D. L.Boger, R. S. Coleman, and B. J. Invergo. J. Org. Chem., 1987, Vol. 52,1521-1530) and triethylamine (0.5 ml, 3.5 mmol) in a mixture ofanhydrous CH₂Cl₂(8 ml) and DMF (2 ml). The mixture was stirred for 20hrs. The resulting clear solution was concentrated in vacuo aid wasadded dropwise to 40 ml of 1M sodium acetate (pH 7.5). The precipitatewas centrifuged, washed with water (2×40 ml), with 10% MeOH in ether(2×40 ml), with ether (40 ml), and with hexane (40 ml). Finally it wasdried in vacuo to give 2f as a pale yellow solid (0.29 g, 91%): (¹H NMR(Me₂SO-d ₆, 200 MHz, ppm) 12.51 (s, 1H, NH′), 11.66 (s, 1H, NH), 8.37(d, 1H, J=8.8 Hz, C4′-H), 8.03 (m, 1H, C₆F₄H), 7.8 (br s, 1H, C4-H),7.58 (s, 1H, C8′-H), 7.40 (d, 1H, J=9.1 Hz, C5′-H), 7.27 (d, 1H, J=8.6Hz, C5-H), 7.1 (s, 1H, C8-H), 4.65 (t, 2H, J=8 Hz, (NCH₂CH₂Y), 4.02 (t,2H, J=9 Hz, NCH₂CH₂), 3.46 (t, 2H, J=8 Hz, NCH₂CH₂)′), 3.25 (t, 2H,J=8.9 Hz, NCH₂CH₂), 1.51 (s, 9H, C(CH₃)₃). Anal. Calcd. forC₃₃H₂₆N₄O₅F₄×0.5H₂O: C, 61.59; H, 4.23; N, 8.71. Found: C, 61.73; H,4.12; N, 8.61.3-carbamoyl-1,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylate trimermethyl ester (3a). A solution of methyl1,2-dihydro-3H-pyrroloindole-7-carboxylate (1.0 g, 5 mmol), 2e (1.2 g,2.1 mmol) and triethylamine (0.1 ml, mmol) in 15 ml of anhydrous DMF wasincubated at RT for 24 hrs and at 0° C. for 12 hrs. The resultinginsoluble solid was collected by filtration, washed with DMF (10 ml),CH₂Cl₂ (20 ml) and ether (20 ml). Drying in vacuo afforded 3a (1.1 g,83%) as a pale yellow solid: (¹H NMR (Me₂SO-d ₆, 200 MHz, ppm) 12.02 (s,1H, NH″), 11.75 (s, 1H, NH′), 11.56 (s, 1H, NH), 8.28 (apparent t, 2H,J=8.3 Hz, C4-H″+C4′-H), 7.98 (d, 1H, J=9.4 Hz, C4-H), 7.98 (d, 1H, J=9Hz, C4-H), 7.39-7.33 (2 d, 2H, C5″-H+C5′-H), 7.23 (d, 1H, J=8.7 Hz,C5-H), 7.14 (d, 1H, J=1.6 Hz, C8″-H), 7.10 (d, 1H, J=1 Hz, C8′-H), 6.97(s, 1H, C8-H), 6.11 (s, 2H, CONH₂), 4.65 (t, 4H, (NCH₂CH₂)″+(NCH₂CH₂)′),3.98 (t, 2H, J=8.7 Hz, NCH₂CH₂), 3.88 (s, 3H, CH₃), 3.48-3.25 (m, 6H,(NCH₂CH₂)″+(NCH₂CH₂)′+NCH₂CH₂ partially obscured with H₂O). Anal. Calcd.for C₃₀H₂₉N₇O₅×4.5H₂O: C, 59.32; H, 5.0; N, 13.03. Found: C, 58.9; N,5.06; N, 13.77.

2,3,5,6-Tetrafluorophenyl 3carbamoyl-1,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylate trimer (3c).2,3,5,6-Tetrafluorophenyltrifluoroacetate (2.*6 g; 10 mmol) was addeddropwise to a suspension of 3b (1.1 g, 1.8 mmol) in 15 ml of anhydrousDMF and triethylamine (1.1 ml, 10 mmol). The mixture was stirred for 3hrs. The mixture was concentrated in vacuo (0.2 mm). The residue wastriturated with a mixture of dry dichloromethane (20 ml) and methanol (2ml). The resulting product was collected by filtration, washed withdichloromethane (20 ml), ether (20 ml), and dried in vacuo to give 1.3 g(95%) of a yellow-green solid: (¹H NMR (Me₂SO-d ₆, 200 MHz, ppm) 12.54(d, 1H, J=1 Hz, NH″), 11.79 (s, 1H, NH′), 11.56 (s, 1H, NH), 8.41 (d,1H, J=9.3 Hz, C4-H″), 8.27 (d, 1H, J=9.4 Hz, C4′-H), 8.03 (m, 1H,C₆F₄H), 7.98 (d, 1H, J=9 Hz, C4-H), 7.56 (s, 1H, C8″-H), 7.45-7.35 (m,2H, C5″-H+C5′-H), 7.23 (d, 1H, J=9.2 Hz, C5-H), 7.13 (s, 1H, C8′-H),6.97 (s, 1H, C8-H), 6.11 (s, 2H, CONH₂), 4.65 (m, 4H,(NCH₂CH₂)″+(NCH₂CH₂)′), 3.98 (t, 2H, J=8.7 Hz, NCH₂CH₂), 3.45 (m, 4H,(NCH₂CH₂)″+(NCH₂CH₂)′), 3.25 (t, 2H, J=8.7 Hz, NCH₂CH₂). Anal. Calcd.for C₄₀H₂₇N₇O₅F₄×2H₂O: C, 61.59; H, 4.23; N, 8.71. Found: C, 61.73; H,4.12; N, 8.61.

[3-carbamoyl-1,2-dihydro-3H-pyrrolo[3,2-c]indole-7-carbox]-1-amido-3-propanoltrimer (3d). A solution of 3-amino-1-propanol (70 μl, 1.4 mmol), 3c (75mg, 0.1 mmol) and triethylamine (0.1 ml, mmol) in 2.5 ml of anhydrousDMF was stirred at RT for 10 hrs. The resulting insoluble solid wascollected by filtration, washed with DMF (2 ml), CH₂Cl₂ (10 ml) andether (20 ml). Drying in vacuo afforded 3d (55 mg, 89%) as a pale yellowsolid: (¹H NMR (Me₂SO-d ₆, 200 MHz, ppm) 11.76 (s, 1H, NH″), 11.65 (s,1H, NH′), 11.57 (s, 1H, NH), 8.47 (m, 1H, C4-H), 8.24 (m, 1H, C4-H),7.99 (d, 1H, J=8.4 Hz, C4-H), 7.40-7.32 (2d, 2H, C8″-H+C5′-H), 7.23 (d,1H, J=8.9 Hz, C5-H), 7.12 (s, 1H, C8″-H), 7.10 (s, 1H, C8′-H), 6.99 (s,1H, C8H), 6.12 (s, 3H, CONH₂+NHCO), 4.66 (t, 4H, (NCH₂CH₂)″+(NCH₂CH₂)′),3.98 (t, 2H, J=8.7 Hz, NCH₂CH₂), 3.51-3.25 (m, 10H,NCH₂CH₂)″+NCH₂CH₂)′+NCH₂CH ₂+CH₂ OH partially obscured with H₂O), 1.70(p, 2H, J=6.6 Hz, CH₂CH₂CH₂).

2,3,5,6-Tetrafluorophenyl3-[N-(9-fluorenylmethoxycarbonyl)]aminopropionate (4).2,3,5,6-Tetrafluorophenyl trifluoroacetate (1.7 g, 6.5 mmol) was addeddropwise to a solution of FMOC-alanine (2.0 g, 6.4 mmol) andtriethylamine (1.0 ml, 7 mmol) in 20 ml of anhydrous CH₂Cl₂. After 1hr., CH₂Cl₂ was removed by evaporation at reduced pressure using rotaryevaporator, redissolved in 30 ml ethylacetate/hexane (1:1). Flashchromatography (4×20 cm, hexane/ethyl acetate, 3:1) afforded rude 4 as awhite solid. It was recrystallized from hexane/ethyl acetate to give 4as a white crystalline solid (2.3 g, 78%): ¹H NMR (CDCl₃, 200 MHz, ppm)7.73 (d, 2H, J=7.1 Hz, aromatic protons), 7.75 (d, 2H, J=7.7 Hz,aromatic protons), 7.24-7.42 (m, 4H, aromatic protons), 7.01 (m, 1H,C₆F₄H), 5.21 (br s, 1H, —CONH—), 4.38 (d, 2H, J=7.1 Hz, —CH₂OCO—), 4.20(m, 1H, benzyl proton), 3.58 (m, −2H, NCH 2), 2.93 (t, 2H, J=5.4 Hz,—CH₂CO—). Anal. Calcd. for C₂₄H₁₇NO₄F₄: C, 62.75; H, 3.73; N, 3.05.Found: C, 62.52; H, 3.59; N, 3.01.

1-[3-[N-(9-Fluorenylmethoxycarbonyl)amino]-1-oxopropyl]amino-(R,S)-2,3-propanediol(5). A solution of 4 (2.0 g, 4.35 mmol) in 20 ml of anhydrous CH₂Cl₂ wasadded to a stirred solution of 3-amino-1,2-propanediol (0.6, mmol) in 10ml MeOH. After 10 min, acetic acid (3 ml) was added and the mixture wasevaporated to dryness. The residue was triturated with 100 ml of water.The obtained solid was filtered off, washed with water and dried byco-evaporation with toluene (2×50 ml) at reduced pressure. Washing with50 ml of ethyl acetate followed by drying in vacuo overnight yielded 5as a white crystalline solid (1.65 g, 99%): ¹H NMR (CDCl₃+MeOD-d4, 200MHz, ppm, Me₄Si) 7.77 (d, 2H, J=7.7 Hz, aromatic protons), 7.61 (d, 2H,J=7.3 Hz, aromatic protons), 7.45-7.29 (m, 4H, aromatic protons), 4.35(d, 2H, J=7.1 Hz, —CH₂OCO—), 4.22 (m, 1H, benzyl proton), 3.72 (m, 1H,—CH— from NHCH₂CHOHCH₂OH), 3.52-3.27 (m, 6H, OCONHCH ₂+CH ₂CHOHCH ₂OH),2.44 (t, 2H, J=6.6 Hz, —CH₂CO—); Anal. Calcd. for C₂₁H₂₄N₂O₅: C, 5.61;H, 6.29; N, 7.29%. Found: C, 65.43; H, 6.28; N, 7.21.

1-[3-[N-(9-Fluorenylmethoxycarbonyl)amino]-1-oxopropyl]amino-(R,S)-2-[[bis(methoxyphenyl)phenylmethoxy]methyl]-2-ethanol(6). To a stirred solution of 5 (1.6 g, 4.2 mmol) in 30 ml of anhydrouspyridine was added DMTrCl (1.6 g, 4.7 mmol). After stirring for 3 hrs.under argon, the mixture was evaporated to dryness. Residual pyridinewas removed by co-evaporation with toluene. The residue was dissolved in100 ml of CH₂Cl₂, washed with 2×100 ml water, dried over sodium sulfate,and evaporated to dryness. The residue was purified by flashchromatography (4×20 cm, silica) using ethyl acetate as an eluent. Thefractions containing pure product were combined and evaporated todryness to yield 1.9 g (66%) of 6 as a colorless foam: ¹H NMR (CDCl₃,200 MHz, ppm, Me₄Si) 7.72 (d, 2H, J=7.2 Hz, aromatic protons), 7.56 (d,2H, J=7 Hz, aromatic protons), 7.40-7.20 (m, 13H, aromatic protons),6.80 (d, 4H, J=9 Hz, DMTr protons), 5.76 (br s, 1H, NH), 5.42 (br s, 1H,NH), 4.35 (d, 2H, J=6.6 Hz, —CH₂OCO—), 4.17 (m, 1H, benzyl proton), 3.83(m, 1H, —CH— from NHCH₂CHOHCH₂OH), 3.75 (s, 6H, OCH₃), 3.60-3.30 (m, 4H,OCONHCH ₂+CH ₂CHOHCH₂OH), 3.13 (d, 2H, J=5.4 Hz, CH₂ODMTr), 2.30 (t, 2H,J=5.4 Hz, —CH₂CO—); Anal. Calcd. for C₄₂H₄₂N₂O₇: C, 73.45; H, 6.16; N,4.08. Found: C, 65.43; H, 6.28; N, 7.21.

2,3,5,6-Tetrafluorophenyl1-[3-[N-(9-fluorenylmethoxycarbonyl)amino]-1-oxopropyl]amino(R,S)-2-[[bis(methoxyphenyl)phenylmethoxy]methyl]-2-ethylbutanedioate (7). To a solution of 6 (1.2 g, 1.75 mmol), triethylamine(0.2 g, 2 mmol), 1-methylimidazole (20 μl) in 10 ml of anhydrous CH₂Cl₂was added 0.2 g (2 mmol) of succinic anhydride. This solution wasstirred for 20 hrs. Triethylamine (60 μl) was added to the solutionfollowed by 0.6 g (2.2 mmol) of 2,3,5,6-tetrafluorophenyltrifluoroacetate. After 1 hr., CH₂Cl₂ was removed by evaporation atreduced pressure using a rotary evaporator, and the residue wasdissolved in 15 ml ethylacetate/hexane (1:2). Flash chromatography (4×20cm, hexane/ethyl acetate, 2:1) afforded 1b as a pale yellow foam (1.2 g,73%): ¹H NMR (CDCl₃, 300 MHz, ppm, Me₄Si) 7.71 (d, 2H, J=7.2 Hz,aromatic protons), 7.54 (d, 2H, J=7, aromatic protons), 7.40-7.20 (m,13H, aromatic protons), 7.00 (m, 1H, C₆F₄H), 6.78 (d, 4H, J=7 Hz, DMTrprotons), 5.71 (br s, 1H, NH), 5.42 (br s, 1H, NH), 5.15 (m, 1H, —CH—from NHCH₂CHOHCH₂OH), 4.31 (d, 2H, J=6.2 Hz, —CH₂OCO—), 4.16 (d, 5.5 Hz,1H, benzyl proton), 3.74 (s, 6H, OCH₃), 3.60-3.30 (m, 4H, OCONHCH ₂+CH₂CHOHCH₂OH), 3.20 (br s, 2H, CH₂ODMTr), 2.98 (br s, 2H, COCH₂CH₂CO),2.72 (br s, 2H, COCH₂CH₂CO), 2.20 (br s, 2H. —CH₂CO—); Anal, Calcd. forC42H₄₂N₂O₇: C, 66.80; H, 4.96; N, 3.00. Found: C, 66.18; H, 4.98; N,2.6.

Preparation of CPG derivative 8. A mixture of 5.0 g of long chainaminoalkyl controlled pore glass (LCAACPG), 0.5 ml of 1-methylimidazole,and 0.45 g (0.5 mmol) of 7 in 20 ml of anhydrous pyridine was swirled in100 ml flask (orbital mixer, 150 rpm). After 3 hrs, the CPG was filteredon a sintered glass funnel and washed with 100 ml portions of DMF,acetone, and diethyl ether. The CPG was dried in vacuo and treated witha mixture of pyridine (20 ml), acetic anhydride (2 ml), and1-methylimidazole (2 ml). After swirling for 30 min, the CPG was washedwith pyridine, methanol, and diethyl ether, then dried in vacuo. Theproduct (8) was analyzed for dimethoxytdtyl (DMTr) content according tothe literature method (T. Atkinson and M. Smith. in M. Gait (ed.),Oligonucleotide Synthesis, A Practical Approach. IRL Press, 1984,Oxford, UK, pp. 35-81) and found to have a loading of 28 μmol/g.

Preparation of CPG derivative 9. The CPG derivative 8 (3.0 g) wastreated twice with 20 ml of 20% piperidine in dry DMF for 5 min eachtime. The CPG was washed with 100 ml portions of DMF, methanol, anddiethyl ether, then dried in vacuo.

Preparation of CPG derivative 10. A mixture of 2.5 g of 9, 7.5 ml oftriethylamine, and 0.38 g (0.5 mmol) of −3c in −7.5 ml of anhydrous DMSOwas swirled in 50 ml flask (orbital mixer, 150 funnel rpm). After 2days, the CPG was filtered on a sintered glass funnel and washed with100 ml portions of DMSO, acetone, and diethyl ether. The CPG was driedin vacuo and treated with a mixture of pyridine (10 ml), aceticanhydride (1 ml), and 1-methylimidazole (1 ml). After swirling for 30min, the CPG was washed with DMSO, pyridine, methanol, and diethylether, then dried in vacuo.

2-[4-(Phenylazo)benzylthio]ethyl5-[(tert-butyloxy)carboxamido]pentylcarboxylate (11).6-[(Tert-butyloxy)carboxamido]hexanoic acid (4.16 g, 18 mmol) was driedby co-evaporation with dry DMF (70° C.). The residue was redissolved indry DMF (25 ml) and 2-[4-(phenylazo)-benzylthio]-ethanol (4.08 g, 15mmol), N,N-dicyclohexyl carbodiimide (3.71 g, 18 mmol),4-dimethylaminopyridine (1.83 g, 15 mmol) were added at 0° C. Afterstirring at 0° C. for 2 h and 20° C. for 12 h, the reaction mixture wasevaporated to dryness by coevaporation with butyl acetate, andadditional ethyl acetate (150 mL) was added. This solution was extractedwith 0.7 M HCl (1×30 mL), 5% NaHCO₃ and H₂O (2×50 mL). The organic layerwas dried over Na₂SO₄ and concentrated with rotary evaporator. Washingwith 20 mL of ether and filtration afforded compound 11 (6.91 g, 89%).¹H NMR (CDCl₃, 200 MHz, ppm): 7.91 (m, 4H), 7.52 (m, 5H), 4.48 (t, 2H),4.34 (s, 2H), 3.20 (t, 2H), 3.08 (m, 2H), 2.35 (t, 2H), 1.64-1.2 (m,7H), 1.41 (s, 9H).

1,2,3-benzotriazol-1-yl1-methyl-4-(tertbutyloxy)carboxamido-pyrrole-2-carboxylate.N,N′-Dicyclohexylcarbodiimide (1.1 g, 5.3 mmol) was added to a solutionof 1-methyl-4-[tert-butyloxy)carboxamido]pyrrole-2-carboxylic acid⁴ (1.2g, 5.2 mmol) and 1-hydroxybenzotriazol (0.63 g, 4.7 mmol). Afterstirring for 1 hr, the mixture was filtered through the sintered glassfilter to separate precipitated N,N′-dicyclohexylcarbodiimide. Thefiltrate was evaporated to dryness, redissolved in a mixture of CHCl₃and pentane (1:1), and loaded onto a silica gel column. The fractionscontaining pure product were combined and evaporated to dryness to give1.45 g (80%) of the desired product as a white solid: mp=138-138.5° C.;¹H NMR (CDCl₃, 200 MHz) 8.04 (d, 1H), 7.49-7.40 (m, 4H), 7.09 (d, 1H),3.87 (s, 3H), 1.50 (s, 9H).

2-[4-(Phenylazo)benzylthio]ethyl5-[1-methyl-4-(tertbutyloxy)carboxamido]pyrrole-2-carboxamido]pentylcarboxylate(12). Trifluoroacetic acid (5 mL) was added at 0° C. to 11 (0.73 g, 1.5mmol). After stirring at 0° C. for 20 min the reaction mixture wasevaporated to dryness by co-evaporation with CHCl₃. The residue wasdissolved in dry CH₂Cl₂ (15 mL) and 1,2,3-benzotriazol-1-yl1-methyl-4-(tert-butyloxy)carboxamido-pyrrole-2-carboxylate (0.59 g,1.65 mmol), dry triethylamine (0.23 g, 2.3 mmol) were added. Afterstirring at ambient temperature for 15 min, CHCl₃ was added (100 mL).The reaction mixture was extracted with 5% NaHCO₃ (2×20 mL), H₂O (2×20mL). The organic layer was dried over Na₂SO₄ and concentrated on arotary evaporator. Chromatography on silica gel (100 g) with CHCl₃afforded 0.88 g (91.8%) 12. ¹H NMR (CDCl₃, 200 MHz, ppm): 7.88 (m, 4H),7.46 (m, 5H), 6.74 (s, 1H), 6.38 (s, 1H), 6.26 (s, 1H), 5.87 (t, 1H),4.18 (t, 2H, J=6 Hz), 3.82 (s, 3H), 3.79 (s, 2H), 3.3 (m, 2H), 2.63 (t,2H, J=6 Hz), 2.30 (t, 2H, J=6 Hz), 1.64-1.2 m, 6H), 1.46 (s, 9H).

2-[4-(Phenylazo)benzylthio]ethyl 5-[1-methyl-4-[1methyl-4-(tert-butyloxy)carboxamidopyrrole-2-carboxamido]pyrrole-2-carboxamido]pentylcarboxylate.(13). A solution of 12 (2.43 g, 4 mmol) in dry CH₂Cl₂ (8 mL) was treatedwith trifluoroacetic acid (4 mL) at 0° C. The resulting solution wasleft at ambient temperature in stopped flask for 1 hr and thenpartitioned between 30% aqueous K₂CO₃ (30 mL) and CH₂Cl₂ (30 mL). Thelower layer was collected. The aqueous phase was extracted withdichloromethane (2×20 mL), and the combined organic extracts, afterbeing washed with H₂O (1×20 mL), were dried over Na₂SO₄ and evaporated.The residue was dissolved in CH₂Cl₂ (10 mL) and 1,2,3-benzotriazol-1-yl1-methyl-4-(tert-butyloxy)carboxamidopyrrole-2-carboxylate (1.43 g, 4mmol) and dry triethylamine (0.8 g, 8 mmol) were added. After stirringat ambient temperature for 30 min, CHCl₃ (100 mL) was added. Thereaction mixture was extracted with 5% NaHCO₃ (2×20 mL), H₂O (2×20 mL).The organic layer and concentrated on a rotary was dried over Na₂SO₄ andconcentrated on a rotary evaporator. Chromatography on silica gel (100g) with CHCl₃ afforded 1.95 g (66.8%) of 13. ¹H NMR (CDCl₃, 200 MHz,ppm): 7.87 (m, 4H), 7.46 (m, 5H), 7.04 (d, 1H, J=1.5 Hz), 6.77 (br s,1H), 6.52 (br s, 1H), 6.50 (d, 1H, J=1.5 Hz), 6.31 (br s, 1H), 5.95 (t,1H), 4.19 (t, 2H, J=6 Hz), 3.85 (s, 6H), 3.78 (s, 2H), 3.32 (m, 2H),2.64 (t, 2H, J=6 Hz), 2.31 (t, 2H, J=6 Hz), 1.64-1.2 (m, 6H) 1.48 (s,9H).

2-[4-(Phenylazo)benzylthio]ethyl5-[1-methyl-4-[1-methyl-4-[1-methyl-4-(tert-butyloxy)carboxamidopyrrole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]pentylcarboxylate(14). A solution of 13 (1.90 g, 2.6 mmol) in dry CH₂Cl₂ (6 mL) wastreated with trifluoroacetic acid (3 mL) at 0° C. The resulting solutionwas left at ambient temperature in stopped flask for 1 hr and thenpartitioned between 30% aqueous K₂CO₃ (30 mL) and CH₂Cl₂ (30 mL). Thelower layer was collected. The aqueous phase was extracted withdichloromethane (2×20 mL), and the combined organic extracts, afterbeing washed with H₂O (1×20 mL), were dried oveNa₂SO₄ and evaporated.The residue was dissolved in CH₂Cl₂ (2.5 mL) and 1,2,3-benzotriazol-1-yl1-methyl-4-(tert-butyloxy)carboxamidopyrrole-2-carboxylate (1.4 g, 3.9mmol), dry triethylamine (0.8 g, 8 mmol) were added. After stirring atambient temperature for 1 h, CHCl₃ (100 mL) was added. The reactionmixture was extracted with 5% NaHCO₃ (2×20 mL), H₂O (2×20 mL). Theorganic layer was dried over Na₂SO₄ and concentrated on a rotaryevaporator. Chromatography on silica gel (100 g) with 0-1.5% methanol inCHCl₃ afforded 1.56 g (70.5%) of 14. ¹H NMR (CDCl₃, 200 MHz, ppm): 7.87(m, 4H), 7.68 (br s, 1H), 7.60 (br s, 1H), 7.46 (m, 5H), 7.08 (br s,2H), 6.78 (br s, 1H), 6.56 (d, 1H, J=1.5 Hz), 6.60 (br s, 1H), 6.55 (d,1H, J=1.5 Hz), 6.03 (t, 1H), 4.18 (t, 2H, J=6 Hz), 3.86 (m, 9H), 3.78(s, 2H), 3.32 (m, 2H), 2.63 (t, 2H, J=6 Hz), 2.30 (t, 2H, J=6 Hz),1.64-1.2 (m, 6H), 1.48 (s, 9H).

2-[4-(Phenylazo)benzylthio]ethyl-5-[1-methyl-4-[(1-methyl-4-[1-methyl-4-[1-methyl-4-(tert-butyloxy)carboxamidopyrrole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]pentylcarboxylate(15). A solution of 14 (0.32 g, 0.32 mmol) in dry CH₂Cl₂ (5 mL) wastreated with trifluoroacetic acid (2.5 mL) at 0° C. The resultingsolution was left at ambient temperature in stopped flask for 1 h andthen partitioned between 30% aqueous K₂CO₃ (30 mL) and CH₂Cl₂ (30 mL).The lower layer was collected. The aqueous phase was extracted withdichloromethane (2×20 mL), and the combined organic extracts, afterbeing washed with H₂O (1×20 mL), were dried over Na₂SO₄ and evaporated.The residue was dissolved in CH₂Cl₂ (1 mL) and 1,2,3-benzotriazol-1-yl1-methyl-4-(tert-butyloxy)carboxamidopyrrole-2-carboxylate (0.11 g, 0.32mmol), dry triethylamine (0.06 g, 0.03 mmol) were added. After stirringat ambient temperature for 1.5 h. CHCl₃ (100 mL) was added. Thesuspension was filtered and the filtrate was extracted with 5% NaHCO₃(2×20 mL), H₂O (2×20 mL). The organic layer was dried over Na₂SO₄ andevaporated to dryness. The yield of 15 was 0.25 g (80%). ¹H NMR (CDCl₃,200 MHz, ppm): 8.17 (br s, 1H), 7.98 (br s), 7.96 (br s), 1H 7.85 (m,4H), 7.44 (m, 5H), 7.09 (br s, 2H), 7.02 (s, 1H), 6.78 (br s, 1H), 6.74(br, 1H), 6.66 (s, 1H), 6.58 (s, 3H), 6.29 (t, 1H), 4.18 (t, 2H, J=6Hz), 3.78 (m, 14H), 3.28 (m, 2H), 2.60 (t, 2H, J=6 Hz), 2.26 (t, 2H, J=6Hz), 1.64-1.2 (m, 6H), 1.48 (s, 9H).

2-[4-(Phenylazo)benzylthio]ethyl5-[1-methyl-4-[1-methyl-4-[1-methyl-4-[1-methyl-4-[1-methyl-4-(tertbutyloxy)carboxamidopyrrole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]pentylcarboxylate(16). A solution of 15 (0.65 g, 0.67 mmol) in dry CH₂ Cl₂ (10 mL) wastreated with trifluoroacetic acid (5 mL) at 0° C. The resultingyellowish solution was left at ambient temperature in stopped flask for1 h and then partitioned between 30% aqueous K₂CO₃ (30 mL) and CH₂Cl₂(30 mL). The lower layer was collected. The aqueous phase was extractedwith dichloromethane (2×20 mL), and the combined organic extracts, afterbeing washed with H₂O (1×20 mL), were dried over Na₂SO₄ and evaporated.The residue was dissolved in DMF (1 mL) and 1,2,3-benzotriazol-1-yl1-methyl-4-(tertbutyloxy)carboxamidopyrrole-2-carboxylate (0.24 g, 0.67mmol), dry triethylamine (0.13 g, 0.67 mmol) were added. After stirringat ambient temperature for 3 h, the reaction mixture was evaporated todryness by coevaporation with butyl acetate. The residue was dissolvedin 3 mL 2.5% DMF in CHCl₃. Chromatography on silica gel (100 g) with0-2.5% methanol in CHCl₃ (2.5% DMF) afforded 0.67 g (45%) of 16.

2,3,5,6-Tetrafluorophenyl-4′-[bis(2-chloroethyl)amino]phenylbutyrate(Chlorambucil 2,3,5,6-tetrafluorophenyl ester)

To a solution of 0.25 g (0.82 mmol) of chlorambucil (supplied by FlukaA. G.), 0.3 g (1.1 mmol) of 2,3,5,6-tetrafluorophenyl trifluoroacetatein 5 ml of dry-dichloromethane was added 0.2 Ml of dry triethylamine.The mixture was stirred Under argon at room temperature for 0.5 h andevaporated. The residual oil was purified by column chromatography onsilica gel with hexane-chloroform (2:1) as the eluting solvent to givethe ester as an oil: 0.28 g (75%); TLC on silica gel (CHCl₃) R_(f) 0.6;IR (in CHCl₃) 3010; 1780, 1613, 1521, 1485 cm⁻¹.

Introduction of Chlorambucil Residue into the Primary Amino Groups ofOligonucleotides.

Preparation of the cetyltrimethylammonium salt of oligonucleotides: a100 μL of aqueous solution of oligonucleotide (50-500 ug), generallytriethylammonium salt, was injected to a column packed with Dowex 50wx8in the, cetyltrimethylammonium form and prewashed with 50% alcohol inwater. The column was eluted by 50% aqueous ethanol (0.1 mL/min).Oligonucleotide containing fraction was dried on a Speedvac over 2 hoursand used in following reactions.

Ethanol solution (50 uL) of cetyltrimethylammonium salt of anoligonucleotide (50-100 μg) was mixed with of 0.08 M solution of2,3,5,6-tetrafluorophenyl-4′-[bis(2-chloroethyl)amino]phenylbutyrate(tetrafluorophenyl ester of chlorambucil) in acetonitrile (50 μL) and 3μL of diisopropylethylamine. After shaking for three hours at roomtemperature, the product was precipitated by 2% LiCl0₄ in acetone (1.5mL). The product was reprecipitated from water (60 uL) by 2% LiClO₄ inacetone three times. Finally, chlorambucil derivative of oligonucleotidewas purified by Reverse Phase Chromatography with approximately 50-80%yield. The fraction containing a product was concentrated byapproximately butanol. Isolated chlorambucil derivative ofoligonucleotide was precipitated in acetone solution of LiClO₄, washedby acetone and dried under vacuum of oil pump. All manipulation ofreactive oligonucleotide was performed as quickly as possible, with theproduct in ice-cold solution, starting from the chromatographic fractioncollected.

Oligonucleotide Synthesis

All oligonucleotides were prepared from 1 mmol of the appropriate CPGsupport on an ABM 394 using protocol supplied by manufacturer. Standardreagents for the β-cyanoethylphosphoramidite coupling chemistry werepurchased from Glen Research. 5′-aminohexyl modifications wereintroduced using an N-MMT-hexanolamine phosphoramidite linker (GlenResearch). 3′-aminohexyl modifications were introduced using the CPGprepared as previously described, C. R. Petrie, M. W. Reed, A. D. Adans,and R. B. Meyer, Jr. Bioconjugate Chemistry, 1992, 3, 85-87.

Preparation of Conjugates (Reaction Scheme 3).

To a solution of cetyltrimethylammonium salt of an aminohexyl modifiedoligonucleotide (30-50 nmol, Jost, J.-P., Jiricny, J., and Saluz, H,(1989) Quantitative precipitation of short oligonucleotides with lowconcentrations of cetyltrimethylammonium bromide. Nucleic Acids Res. 17,2143) and 1.5 μl of N,N-diisopropylethylamine in 40 μl of dry DMSO wasadded 40 μl of 4 mM solution of the TFP ester (1a, 1b, 2e, 2f or 3c).The reaction mixture was kept for 12 hrs at RT. The oligonucleotiderelated material was precipitated by addition of 1.5 ml of 2% LiCl₄ inacetone. The pellet was washed with acetone, and dried in vacuo. Thepellet was redissolved in 60 μl of 50% DMF in H₂O and precipitated againas described above using 2% solution of LiClO₄ in acetone. Thisprocedure was repeated twice. The residue was purified by HPLC (4.6×250mm, C-18, Dynamax-300A, Rainin) using a gradient of acetonitrile from 20to 75% in the presence of 50 mM LiClO₄. The fraction containing pureproduct was dried in vacuo using speedvac. The residue was dissolved in60-80 μl of H₂O and precipitated with 1.5 ml of 2% LiClO₄ in acetone.After washing with acetone (2×1.5 ml) and drying in vacuo, the pelletwas dissolved in 100 μl of H₂O. The yield of final product was 20-50%.

A modified procedure of Godovikova et al. (T. S. Godovikova, V. F.Zarytova, T. V. Maltzeva, L. M. Khalimskaya. Bioorgan. Khim., 1989, 15,1246-1259) was used for the preparation of the oligonucleotideconjugates bearing 4-amino-N-methylpyrrol-2-carboxylic acid residues. Asolution of cetyltrimethylammonium salt of 3′-phosphate-containingoligonucleotide (50-100 nmol), triphenylphospine (10 mg),2,2′-dipyridyldisulfide (10 mg), N,N-dimethylaminopyridine (10 mg), andone of the analogues selected from compounds 11 through 16 in 100 μl ofdry DMF was incubated for 20 min at RT. The oligonucleotide relatedmaterial was precipitated by addition of 1.5 ml of 2% LiclO₄ in acetone.The pellet was washed with acetone, and dried in vacuo. The residue waspurified by HPLC using gradient of acetonitrile from 20 to 75% inpresence of 50 mM LiClO₄. The fraction containing pure product was driedin vacuo using speedvac. The residue was dissolved in 60-80 μl of H₂Oand precipitated with 1.5 ml of 2% LiClO₄ in acetone. After washing withacetone (2×1.5 ml) and drying in vacuo, the pellet was dissolved in 100μl of H₂O. The yield of final product was 30-50%.

Preparation of Conjugates (Reaction Scheme 4).

CPG containing 5′-aminohexyl derivatized oligonucleotide obtained in asynthesis on 1 μmol scale was treated with 2% dichloroacetic acid inCH₂Cl₂ to remove the 9-fluorenylmethoxycarbonyl (Fmoc) protecting groupfrom the amino group followed by washing with acetonitrile, and dryingby flushing with argon. The CPG was transferred into 1.5 ml plastic tubeand 100 ul of 50 mM solution of TFP ester in anhydrous DMSO was added.The tube was shaken for 24 hrs, then washed with 3×1.5 ml DMSO, 2×1.5 mlacetone, and dried in vacuo. The CPG was treated with concentratedammonia to deprotect oligonucleotide using standard conditions. Theresulting reaction mixture was separated using reverse phase HPLC asdescribed above. Typical yield was about 50%.

Thermal Denaturation Studies.

Optical melting curves of oligonucleotide complexes bearing4-amino-N-methylpyrrol-2-carboxylic acid residues were obtained in 200mM NaCl, 10 mM Na₂HP0₄, 0.1 mM EDTA (pH 7.0) on the UV detector of aMilichrom liquid chromatograph in a thermoregulated cell speciallydesigned for this purpose. The data were collected and processed on apersonal computer as described by S. G. Lokhov et al. (S. G. Lokhov, M.A. Podyminogin, D. S. Sergeev, V. M. Silnikov, I. V. Kutyavin, G. V.Shishkin, V. F. Zarytova. Bioconjugate Chem. 1992, 3, 414).

The oligonucleotide complexes carrying1,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylic acid (CDPI) residueswere melted in 140 mM KCl, 10 mM MgCl₂, 20 mM HEPES-HCL (pH 7.2) on aLambda 2 (Perkin Elmer) spectrophotometer with a PTP-6 automaticmulticell temperature programmer. The melting temperatures of complexes(Tm) were determined from the derivative maxima.

Primer Extension Reactions

Primer extension reactions were performed as previously described byLee, et al, (1994) Biochemistry 33: 6024-6030. The final concentrationsof template, primer and blocking ODMs were 5×10⁻¹⁰ M, 4×10⁻⁸ M and 10⁻⁹M, respectively. Primer extension was carried out for 15 min at 45° C.and the products were analyzed by denaturing gel electrophoresis asdescribed in the reference.

In the absence of any blocking ODN, the primer extension reactiongenerated a high molecular weight product which ran as an unresolvedband in the sequencing gel. Weak bands corresponding to pause sites orto spontaneous termination events were reproducibly observed in allreaction mixtures. Unmodified 16-mer and 32-mer ODNS, fullycomplementary to the target, failed to block primer extension. Alsowithout activity were complementary 8-mer and 16-mer ODNS, each of whichwas 3′-linked to a CDPI₃ group. Only a fully complementary 16-mer ODNwith a 5′-conjugated CDPI₃ group arrested primer extension by T7 DNApolymerase. A complementary 8-mer ODN with the same 5′ modificationgenerated only a trace amount of blocked product. Control ODNs confirmedthat inhibition of primer extension required both a complementary ODNand a covalently linked MGB. Two singly-mismatched 16-mer ODNS, eachwith a 5′-linked CDPI₃ peptide, were much less inhibitory than theperfectly matched ODN-MGB conjugates. Addition of unmodified 16-mer ODNtogether with an equimolar amount of free CDPI₃ had no effect on primerextension, emphasizing the importance of the conjugation of the NGB tothe ODN. When a 5′ acridine moiety was conjugated to the fullycomplementary 16-mer ODN instead of the MGB, a loss of inhibitoryactivity was seen.

Cell Culture Crosslinking Experiment

The ODN-MGB conjugate was complementary to nucleotides 815-864 of thetemplate strand of the DQβ1 allele. Proc. Natl. Acad. Sci. USA (1983)80: 7313-7317. The human BSM B-cells used here are homozygous for thisallele and express it constitutively. Prior to adding the ODN, the BSMcells were grown in a 25 ml flask to a density of 4.5×10⁶ cells per mlof media.

For each treatment the cells from a 2 ml aliquot of culture werepelleted and resuspended in 200 ul of serum free media which contained0, 1, 10 or 50 μM 50-mer chlorambucil-linked ODN (either with or withouta 3′ conjugated CDPI₃ group). Each sample was incubated for 3.5 hours at37° C. with 5% CO₂ in a 48-well microtiter plate. The cells were thentransferred to Eppendorf 0.5 ml centrifuge tubes, pelleted 5 min at2,000 rpm, washed twice with 500 μl phosphate buffered saline (PBS) anddeproteinized with Proteinase K/SDS overnight at 370° C. Afterphenol/chloroform extraction and Rnase A digestion the DNA was treatedwith 1M pyrrolidine at 90° C. for 30 min. Pyrrolidine was removed byethanol precipitation, and the ligation-mediated polymerase chainreaction (PCR) reaction was performed as described by Lee et al., supra.Amplified DNA was analyzed on a sequencing gel to visualize any sequencespecific nicking that might have resulted from alkylation of the targetby the chlorambucil-containing ODNS. Results showed cleavage at thenucleotide on the target adjacent to the crosslinker on the ODN, andthat the CDPI₃-containing 50-mer was 10-fold more efficient than thesame ODN without the MGB in sequence specifically alkylating the 0302allele.

Complete media was prepared from the following components (the serumfree media lacked HI-FCS): 500 ml RPMI 1640 with L-Glutamine (2 mM)(Gibco BRL Cat. No. 11875-036)

50 ml of HI-FCS (Gibco BRL Cat. No. 26140, heat inactivated 30 min at55° C.) 5 ml of 100× Penn/Strep (Gibco BRL Cat. No. 15070-022) 5 ml of200 mM L-Glutamine (Gibco BRL Cat. No. 25030-024)

5 ml of 100× Sodium Pyruvate (11 mg/ml; made from Gibco BRL Cat. No.11840-030)

5 ml of 1 M HEPES, pH 7.3 (Gibco BRL Cat. No. 15630023)

Improved Hybridization and Discriminatory Properties ofMGB-Oligonucleotide Conjugates

In many types of hybridization assay, base-pairing interactions betweena probe oligonucleotide and a fully- or partially-complementary targetsequence are detected, either directly (by measuring hybridized probe)or indirectly (by measuring some event that depends on probehybridization). Modifications which improve hybridization kinetics(i.e., speed up the hybridization process), change the equilibrium ofthe hybridization reaction to favor product (i.e., increase the fractionof probe in hybrid), and/or lead to the formation of more stablehybrids, will allow more rapid, efficient and accurate hybridizationassays, thereby increasing efficiency of hybridization and facilitatingthe use of hybridization techniques in new areas such as diagnostics andforensics. Furthermore, it is often advantageous to be able todistinguish between a perfect hybrid (or perfect match), in which everyprobe nucleotide is base-paired to a complementary nucleotide in thetarget, and an imperfect hybrid or mismatch, in which one or more probenucleotides are not complementary to the target. For example, a hybridbetween an oligonucleotide and a target nucleic sequence wherein onebase in the oligonucleotide is non-complementary to the target sequenceis termed a single-nucleotide mismatch. Single-nucleotide mismatchdiscrimination (i.e., the ability to distinguish between a perfect matchand a single-nucleotide mismatch) is extremely useful in the detectionof mutations for diagnostic purposes, and in the determination ofallelic single-nucleotide polymorphisms in diagnostic, therapeutic, andforensic applications. Heretofore, such single-nucleotide mismatchdiscrimination has been difficult to achieve, especially underconditions of high stringency.

The present invention provides, among other things, modifiedoligonucleotides for use as probes and primers. The oligonucleotides aremodified by the covalent attachment of a minor groove binding moiety(MGB). The structure and preparation of exemplary MGBs are providedherein. A MGB-oligonucleotide conjugate having a defined sequence thatis complementary to a target sequence in a second polynucleotide willform a duplex having greater hybrid strength, compared tooligonucleotide probes and primers of the prior art. AMGB-oligonucleotide conjugate whose sequence will result in a hybridhaving a single-nucleotide mismatch with that of a target sequence in asecond polynucleotide will form a duplex that is more easilydiscriminated from a perfectly-matched duplex, compared tooligonucleotide probes and primers of the prior art.

Increased Hybrid Stability

The strength of hybridization, or hybrid stability, between two nucleicacids, can be determined by subjecting a nucleic acid duplex to steadilyincreasing temperature or to a steadily increasing concentration of adenaturing agent. Ultraviolet absorbance (a measure of base-pairing) isdetermined as a function of temperature or concentration of denaturingagent. Absorbance increases as base pairs become unpaired and basestacking interactions are lost, and absorbance reaches a plateau whenthe duplex has been completely denatured. Several measures ofhybridization strength can be obtained from this type of analysis. Themelting temperature (T_(m)) is commonly defined as the temperature (orconcentration) at which half of the base-pairs in the duplex becomeunpaired. The temperature at which the change in absorbance with respectto temperature (or with respect to the concentration or intensity ofdenaturing agent) is at a maximum (i.e., the temperature at which dA/dTis at a maximum) is known as the T_(max) for that particular nucleicacid duplex.

Increased hybridization strength between MGB-oligonucleotide conjugatesand their target sequences has several advantageous consequences. Forexample, short oligonucleotides, less than about 21 nucleotides inlength, are generally not suitable for use in amplification techniquessuch as PCR, because such techniques are conducted at elevatedtemperatures (usually over 70° C.) that are above the T_(m) of shortoligonucleotides. However, conjugation of a MGB to such a shortoligonucleotide results in an increase in hybrid stability sufficientfor the MGB-oligonucleotide conjugate to achieve specific, stablehybridization at elevated temperatures, such as are used in PCR andother amplification techniques.

Another advantage of the increased hybrid stability conferred onoligonucleotides by MGB conjugation is that it is now possible to obtainhybridization to a target using an oligonucleotide containing one ormore complementary sequence mismatches with the target sequence. Thiswill make it possible to use a single defined sequence oligonucleotideas a primer or probe for a target that exhibits genetic heterogeneity.To provide just one example, MGB-oligonucleotide conjugates are usefulin assays which utilize oligonucleotides for the detection of HIV. Sincethe HIV genome undergoes frequent mutational events, some of which leadto resistance to anti-viral therapeutics, it is possible that a mutationpresent in a region of the viral genome complementary to anoligonucleotide probe or primer will result in the mutant becomingundetectable in an oligonucleotide-based assay. However,MGB-oligonucleotide conjugates, by virtue of their increased hybridstability, allow an oligonucleotide with one or more nucleotidemismatches to recognize a specific target sequence, thereby functioningas an effective probe and/or primer in systems characterized by atendency toward frequent mutation of the target sequence. In addition,multiple subtypes of a virus, distinguished by changes in one or a fewnucleotides in the target region, can be detected with aMGB-oligonucleotide conjugate having a single sequence.

Although a MGB-conjugated oligonucleotide is stabilized in its duplexrelative to a non-conjugated oligonucleotide, the conjugated oligomaintains its sequence specificity, under appropriate conditions ofstringency, such that mismatches can be discriminated (i.e., thestability of a duplex formed by a perfectly-matched oligonucleotide ishigher than that of a mismatched duplex when the oligonucleotide isconjugated to a MGB, even though the T_(m) of both duplexes is increasedby MGB conjugation). That is to say, if a perfectly-matchedoligonucleotide and an oligonucleotide differing by one nucleotide werecompared (in terms of the strength of hybrids formed with a specifictarget sequence), the T_(m) of hybrids formed by both oligonucleotideswould increase, but the T_(m) of the hybrid formed by theperfectly-matched oligonucleotide would increase by a greater extent.This effect is particularly pronounced with shorter oligonucleotides,preferably those shorter than 21 nucleotides in length and, mostpreferably, those shorter than 11 nucleotides in length. The result isthat, even though mismatched probes form more stable hybrids whenconjugated to a MGB, it is possible to distinguish a perfect match froma single-nucleotide mismatch using a MGB-oligonucleotide conjugate. Infact, the difference in T_(m) between a perfect match and asingle-nucleotide mismatch is heightened when MGB-oligonucleotideconjugates are used as probes and/or primers (see infra).

As described herein, the present inventors have discovered that covalentattachment of a MGB to an oligonucleotide dramatically increases thestability of hybrids formed by that oligonucleotide, as measured, forexample, by an increase in T_(m) of the hybrid. Increased hybridstability for short oligonucleotides, as provided by the invention,provides short oligonucleotides useful as primers in various proceduresinvolving primer extension, such as PCR, provided the appended MGB doesnot block the 3′-end of the oligonucleotide in a way that inhibitsextension. It is shown herein (see, Example 1, infra) thatMGB-oligonucleotide conjugates are indeed capable of being extended fromtheir 3′-end by a polymerase. Thus, very short oligonucleotides (lessthan about 21-mers), which of themselves would form fairly unstablehybrids, are able, when conjugated to a MGB, to form hybrids with theirtarget sequence that are stable enough to serve as primers inamplification reactions such as PCR.

There are several advantages to the use of short oligonucleotides asprimers in amplification processes such as PCR. These advantages havenot previously been available in procedures which are normally conductedat high temperatures using thermophilic enzymes. For example, shorteroligonucleotides are easier and less expensive to produce. Additionally,more rapid hybridization kinetics may be obtained with shortoligonucleotides. Furthermore, homology searches conducted using PCR orrelated priming and/or amplification techniques can be based on veryshort regions of homology, on the order of 2-3 codons. Within this sizerange, single-nucleotide mismatch discrimination is maintained byMGB-oligonucleotide conjugates (see infra), making possible verysensitive searches over very limited regions of homology.

Enhanced Mismatch Discrimination

It is becoming increasingly important to be able to distinguish twosequences which differ from each other by a single nucleotide.Single-nucleotide polymorphisms form the molecular basis of manydiseases, can determine an individual's response to a particulartherapeutic, and are useful from a forensic viewpoint. The ability todiscriminate between two polynucleotides which differ by a singlenucleotide is an important and valuable property of MGB-oligonucleotideconjugates. Conjugates between a MGB and a short (<21-mer)oligonucleotide retain the sequence specificity and discriminatoryproperties of short oligonucleotides while, at the same time, beingcapable of forming hybrids having stability characteristics of longeroligonucleotides. Without wishing to be bound by any particular theory,a possible explanation for the combination of these desirable propertiesin a MGB-oligonucleotide conjugate is as follows. Since each base-paircontributes to the stability of a hybrid; the shorter the hybrid, thegreater the relative contribution of each individual base pair to thestability of a probe-target hybrid. Hence, the shorter the probe, thegreater the difference in stability between a probe forming a perfectmatch with its target and a probe having a single base mismatch. Thus,all other things being equal, the shorter the oligonucleotide, thegreater its ability to discriminate between a perfect match and a singlenucleotide mismatch. However, a short oligonucleotide (even one thatforms a perfect match with its target) is not able to form a stablehybrid at the elevated temperatures normally used in amplificationtechniques such as PCR. Thus, the potential discriminatory power ofshort oligonucleotides cannot be exploited in PCR and relatedamplification methods.

One of the effects of conjugating a MGB to a short oligonucleotide is toincrease the stability (and hence the T_(m)) of a hybrid involving theshort oligonucleotide to a degree that is compatible with use of theoligonucleotide at the elevated temperatures necessary for amplificationreactions involving thermostable polymerizing enzymes. At the same time,the discriminatory properties (i.e. the heightened difference in T_(m)between a perfect hybrid and a single base mismatch) characteristic ofshort oligonucleotides are retained by the MGB-oligonucleotide conjugateunder these conditions. See Examples 2, 3 and 6, infra.

Additional Advantages

Since kinetics of hybridization are inversely proportional to the lengthof an oligonucleotide, another useful property of theMGB-oligonucleotide conjugates is that they are capable of annealing totheir target more rapidly than are longer oligonucleotides. Anadditional factor leading to more rapid hybridization kinetics ofMGB-oligonucleotide conjugates is that the MGB is likely to serve as anucleation site for hybridization. Furthermore, the additional freeenergy of binding imparted by the MGB most likely lowers the off-rate ofbinding between the oligonucleotide and its target. As a result,hybridization assays employing MGB-oligonucleotide conjugates can beconducted more rapidly than assays employing longer unconjugatedoligonucleotides that form hybrids of comparable stability. Hence, anadditional advantage provided by the novel compositions of the inventionis that it is now feasible to perform hybridization analyses insituations in which time is limited. To provide but one example, onecould use the methods and compositions of the invention in a surgicalprocedure, to investigate the molecular properties of excised tissue.Combining several advantageous properties of MGB-oligonucleotideconjugates, one would be able to test for molecular propertiescharacteristic of the transformed state (for example, single-basemutations in oncogenes or tumor suppressor genes) in cells from aportion of resected tissue, during surgery, to guide the extent ofresection. A description of the use of the methods and compositions ofthe invention in various techniques of mutation detection is providedherein.

Exemplary Applications

The methods and compositions of the present invention can be used with avariety of techniques, both currently in use and to be developed, inwhich hybridization of an oligonucleotide to another nucleic acid isinvolved. These include, but are not limited to, techniques in whichhybridization of an oligonucleotide to a target nucleic acid is theendpoint; techniques in which hybridization of one or moreoligonucleotides to a target nucleic acid precedes one or morepolymerase-mediated elongation steps which use the oligonucleotide as aprimer and the target nucleic acid as a template; techniques in whichhybridization of an oligonucleotide to a target nucleic acid is used toblock extension of another primer; techniques in which hybridization ofan oligonucleotide to a target nucleic acid is followed by hydrolysis ofthe oligonucleotide to release an attached label; and techniques inwhich two or more oligonucleotides are hybridized to a target nucleicacid and interactions between the multiple oligonucleotides aremeasured. Conditions for hybridization of oligonucleotides, and factorswhich influence the degree and specificity of hybridization, such astemperature, ionic strength and solvent composition, are well-known tothose of skill in the art. See, for example, Sambrook et al., supra;Ausubel, et al., supra; M. A. Innis et al (eds.) PCR Protocols, AcademicPress, San Diego, 1990; B. D. Hames et al. (eds.) Nucleic AcidHybridisation: A Practical Approach, IRL Press, Oxford, 1985; and vanNess et al. (1991) Nucleic Acids Res. 19:5143-5151.

Hybridization Probes

In one aspect of the present invention, one or more MGB-oligonucleotideconjugates can be used as probe(s) to identify a target nucleic acid byassaying hybridization between the probe(s) and the target nucleic acid.A probe may be labeled with any detectable label, or it may have thecapacity to become labeled either before or after hybridization, such asby containing a reactive group capable of association with a label or bybeing capable of hybridizing to a secondary labeled probe, either beforeor after hybridization to the target. Conditions for hybridization ofnucleic acid probes are well-known to those of skill in the art. See,for example, Sambrook et al., supra; Ausubel et al., supra; Innis etal., supra; Hames et al. supra; and van Ness et al., supra.

Hybridization can be assayed (i.e., hybridized nucleic acids can beidentified) by distinguishing hybridized probe from free probe by one ofseveral methods that are well-known to those of skill in the art. Theseinclude, but are not limited to, attachment of target nucleic acid to asolid support, either directly or indirectly (by hybridization to asecond, support-bound probe or interaction between surface-bound andprobe-conjugated ligands) followed by direct or indirect hybridizationwith probe, and washing to remove unhybridized probe; determination ofnuclease resistance; buoyant density determination; affinity methodsspecific for nucleic acid duplexes (e.g., hydroxyapatitechromatography); interactions between multiple probes hybridized to thesame target nucleic acid; etc. See, for example, Falkow et al., U.S.Pat. No. 4,358,535; Urdea et al., U.S. Pat. Nos. 4,868,105 and5,124,246; Freifelder, Physical Biochemistry, Second Edition, W. H.Freeman & Co., San Francisco, 1982; Sambrook, et al., supra; Ausubel etal, supra; Hames et al., supra; and other related references. Theduplex-stabilizing capability of MGB-oligonucleotide conjugates makeshybridization possible under more stringent conditions, whereinpotentially occluding secondary structure in the target nucleic acid canbe minimized.

Amplification Primers

Amplification procedures are those in which many copies of a targetnucleic acid sequence are generated, usually in an exponential fashion,by sequential polymerization and/or ligation reactions. Manyamplification reactions, such as PCR, utilize reiterativeprimer-dependent polymerization reactions. A primer is a nucleic acidthat is capable of hybridizing to a second, template nucleic acid andthat, once hybridized, is capable of being extended by a polymerizingenzyme (in the presence of nucleotide substrates), using the secondnucleic acid as a template. Polymerizing enzymes include, but are notlimited to, DNA and RNA polymerases and reverse transcriptases; etc.Conditions favorable for polymerization by different polymerizingenzymes are well-known to those of skill in the art. See, for example.Sambrook et al., supra; Ausubel, et al., supra; Innis et al., supra.Generally, in order to be extendible by a polymerizing enzyme, a primermust have an unblocked 3′-end, preferably a free 3′ hydroxyl group. Theproduct of an amplification reaction is an extended primer, wherein theprimer has been extended by a polymerizing enzyme.

Thus, in one embodiment of the invention, the methods and compositionsdisclosed and claimed herein are useful in improved amplificationreactions such as PCR. See, e.g., U.S. Pat. Nos. 4,683,202; 4,683,195and 4,800,159; Mullis and Faloona, supra; and Saiki et al., supra. Thepolymerization step of PCR is most often catalyzed by a thermostablepolymerizing enzyme, such as a DNA polymerase isolated from athermophilic bacterium, because of the elevated temperatures requiredfor the denaturation step of PCR. As discussed supra, one of theproblems heretofore associated with the practice of PCR is therequirement for relatively long oligonucleotide primers, havingsufficient hybrid stability to serve as primers at the elevatedtemperatures under which PCR is conducted. MGB-oligonucleotideconjugates are useful as primers in amplification reactions such as PCR,since conjugation of a MGB to an oligonucleotide increases hybridstability, thereby significantly extending the lower limit of usefulprimer length. In addition, MGB-oligonucleotide conjugates are useful inspecialized PCR protocols wherein reduced primer length is desirable.These include, but are not limited to, differential display, in whichoptimal primer length is below 10 nucleotides, random amplification ofpolymorphism in DNA (RAPD) techniques, and amplification lengthpolymorphism analyses. Liang et al, supra; Williams et al., supra.

The improvements provided by the present invention are applicable to anytype of assay or procedure in which PCR or a related amplificationtechnique is used, including, but not limited to, hydrolyzable probeassays, priming with allele-specific oligonucleotides (ASOs), fragmentlength polymorphism analysis, single nucleotide polymorphism (SNP)analysis and microsatellite analysis, for example. These and othertechniques are useful in gene mapping, in the identification andscreening of disease-related genes, and in pharmacogenetics, to namejust a few applications.

Assays Utilizing Labeled Probes; Including Hydrolyzable Probe Assays

Additional uses for MGB-oligonucleotide conjugates are found in assaysin which a labeled probe is hybridized to a target and/or an extensionproduct of a target, and a change in the physical state of the label iseffected as a consequence of hybridization. A probe is a nucleic acidmolecule that is capable of hybridizing to a target sequence in a secondnucleic acid molecule. By way of example, one assay of this type, thehydrolyzable probe assay, takes advantage of the fact that manypolymerizing enzymes, such as DNA polymerases, possess intrinsic 5′-3′exonucleolytic activities. Accordingly, if a probe is hybridized to asequence that can serve as a template for polymerization (for instance,if a probe is hybridized to a region of DNA located between twoamplification primers, during the course of an amplification reaction),a polymerizing enzyme that has initiated polymerization at an upstreamamplification primer is capable of exonucleolytically digesting theprobe. Any label attached to such a probe will be released, if the probeis hybridized to its target and if amplification is occurring across theregion to which the probe is hybridized. Released label is separatedfrom labeled probe and detected by methods well-known to those of skillin the art, depending on the nature of the label. For example,radioactively labeled fragments can be separated by thin-layerchromatography and detected by autoradiography; whilefluorescently-labeled fragments can be detected by irradiation at theappropriate excitation wavelengths with observation at the appropriateemission wavelengths. See, e.g., U.S. Pat. No. 5,210,015.

In a variation of this technique, a probe contains both a fluorescentlabel and a quenching agent, which quenches the fluorescence emission ofthe fluorescent label. In this case, the fluorescent label is notdetectable until its spatial relationship to the quenching agent hasbeen altered, for example by exonucleolytic release of the fluorescentlabel from the probe. Thus, prior to hybridization to its targetsequence, the dual fluorophore/quencher labeled probe does not emitfluorescence. Subsequent to hybridization of thefluorophore/quencher-labeled probe to its target, it becomes a substratefor the exonucleolytic activity of a polymerizing enzyme which hasinitiated polymerization at an upstream primer. Exonucleolyticdegradation of the probe releases the fluorescent label from the probe,and hence from the vicinity of the quenching agent, allowing detectionof a fluorescent signal upon irradiation at the appropriate excitationwavelengths. This method has the advantage that released label does nothave to be separated from intact probe. Multiplex approaches utilizemultiple probes, each of which is complementary to a different targetsequence and carries a distinguishable label, allowing the assay ofseveral target sequences simultaneously.

The use of MGB-oligonucleotide conjugates in this and related methodsallows greater speed, sensitivity and discriminatory power to be appliedto these assays. In particular, the enhanced ability ofMGB-oligonucleotide conjugates to allow discrimination between a perfecthybrid and a hybrid containing a single-base mismatch will facilitatethe use of hydrolyzable probe assays in the identification ofsingle-nucleotide polymorphisms and the like. Examples 2 and 3, infra,provide several examples of the utility of MGB-oligonucleotideconjugates in this type of assay. It will be clear to those of skill inthe art that compositions and methods, such as those of the invention,that are capable of discriminating single-nucleotide mismatches willalso be capable of discriminating between sequences that have multiplemismatches with respect to one another.

Fluorescence Energy Transfer

In further embodiments of the invention, MGB-oligonucleotide conjugatescan be used in various techniques which involve multiplefluorescently-labeled probes. In some of these assays, fluorescenceand/or changes in properties of a fluorescent label are used to monitorhybridization. For example, fluorescence resonance energy transfer(FRET) has been used as an indicator of oligonucleotide hybridization.In one embodiment of this technique, two probes are used, eachcontaining a different fluorescent label. One of the labels is afluorescence donor, and the other is a fluorescence acceptor, whereinthe emission wavelengths of the fluorescence donor overlap theabsorption wavelengths of the fluorescence acceptor. The sequences ofthe probes are selected so that they hybridize to adjacent regions of atarget nucleic acid, thereby bringing the fluorescence donor and thefluorescence acceptor into close proximity, if target is present. In thepresence of target nucleic acid, irradiation at wavelengthscorresponding to the absorption wavelengths of the fluorescence donorwill result in emission from the fluorescence acceptor. These types ofassays have the advantage that they are homogeneous assays, providing apositive signal without the necessity of removing unreacted probe. Forfurther details and additional examples, see, for example, EuropeanPatent Publication 070685; and Cardullo, et al. (1988) Proc. Natl. Acad.Sci. USA 85: 8790-8794. Additional embodiments of the present inventionwill be found in these and related techniques in which interactionsbetween two different oligonucleotides that are hybridized to the sametarget nucleic acid are measured. The selection of appropriatefluorescence donor/fluorescence acceptor pairs will be apparent to oneof skill in the art, based on the principle that, for a given pair, theemission wavelengths of the fluorescence donor will overlap theabsorption wavelengths of the fluorescence acceptor. The enhancedability of MGB-oligonucleotide conjugates to distinguish perfect hybridsfrom hybrids containing a single base mismatch facilitates the use ofFRET-based techniques in the identification of single-nucleotidepolymorphisms and the like.

Use of MGB-Oligonucleotide Conjugates in Assays Involving FluorescenceQuenching

In a further embodiment of the invention, MGB-oligonucleotide conjugatesare useful in assays which utilize the principles of fluorescencequenching. In one version of this type of assay, the principles offluorescence quenching are combined with those of hydrolyzable probes,as discussed supra. In this case, an oligonucleotide probe contains afluorescent label at one end (usually the 5′-end) and a quenching agentat the opposite end (usually the 3′-end). Exemplary fluorescent labelsinclude, but are not limited to, fluoresceins, cyanines, rhodamines andphycoerythrins. Exemplary quenching agents include, but are not limitedto, rhodamines, including tetramethylrhodamine (TAMRA), and compoundcapable of absorbing UV or visible light. The preferred labels arefluorescein and its derivatives and preferred quenching agents arerhodamine derivatives, particularly TAMRA. When the probe is free insolution or hybridized to its target, irradiation of the fluorophore atthe appropriate excitation wavelengths fails to cause fluorescentemission, due to the proximity of the quenching agent to the fluorophoreon the oligonucleotide. However, if probe, that has hybridized to itstarget, is subjected to exonucleolytic hydrolysis by a polymerizingenzyme that has initiated polymerization at an upstream primer, thefluorophore will be released from the oligonucleotide. Subsequent to itsrelease from the oligonucleotide, the fluorophore will be capable offluorescing upon excitation at the appropriate wavelengths, since it hasbeen released from the vicinity of the quencher. MGB-oligonucleotideconjugates, by enhancing the ability to distinguish perfect hybrids fromhybrids containing a single base mismatch, facilitate the use ofoligonucleotides containing fluorophore/quencher combinations in theidentification of single-nucleotide polymorphisms and the like.Exemplary oligonucleotides for use in this aspect of the inventioncontain a conjugated fluorophore, a conjugated quencher and a conjugatedMGB. This type of assay is becoming increasingly important, especiallyin clinical applications, because it is a homogeneous assay (i.e., noproduct separation steps are required for analysis) in which the resultscan be monitored in real time. See, for example, Wittwer et al. (1997)BioTechniques 22:130-138. Rapid, fluorescence-based molecular assaysfind use in, for example, real-time surgical and therapeuticapplications, as well.

Additional assays involving the principles of fluorescence quenchingwill be apparent to those skilled in the art, as will the advantages ofusing MGB-oligonucleotide conjugates in such assays. It will also beclear to those of skill in the art that fluorescently-labeledMGB-oligonucleotide conjugates provide improvements in speed,sensitivity, specificity and discriminatory power in the practice of alltypes of hybridization assays.

PCR Clamping

As described herein, the ability of a MGB-oligonucleotide conjugate (inwhich the MGB is conjugated to the 5′-end of the oligonucleotide) toblock elongation from an upstream primer demonstrates thatMGB-oligonucleotide conjugates find use and provide improvements intechniques such as PCR clamping. See, for example, Giovannangeli et al.(1993) Proc. Natl. Acad. Sci. USA 90:10013-10017. Additionalmodifications of MGB-oligonucleotide conjugates as described Infra, suchas inclusion of phosphorothioate or other modified internucleotidelinkages at the 5′-end of the oligonucleotide, will further increase theusefulness of the compositions of the invention in techniques such asPCR clamping.

Assays Involving Oligonucleotide Ligation

MGB-oligonucleotide conjugates are useful in assays in which two or moreoligonucleotides, complementary to adjacent sites on a target nucleicacid, are hybridized to adjacent sites on the target nucleic acid andligated to one another. See, for example, European Patent Publication320,308; European Patent Publication 336,731; and U.S. Pat. No.4,883,750. Conditions for ligation are well-known to those of skill inthe art. See, for example, Sambrook et al., supra; Ausubel, et al.,supra; Innis et al., supra. Ligated nucleic acids can be identified, forexample, by an increase in size of the product compared to the startingoligonucleotides. The ability to use shorter oligonucleotides in thesetypes of ligation assay enables smaller, more precise regions ofsequence to be probed, which is especially useful in assays based onshort regions of homology. Also, as in the case with hybridizationassays, use of MGB-oligonucleotide conjugates in ligation assaysinvolving ligation of oligonucleotides allows more efficientdiscrimination between perfect hybrids and single-base mismatches, whichis especially important in oligonucleotide ligation assays. Furthermore,ligation assays often have very narrow temperature optima. The abilityof MGB conjugation to raise the T_(m) of an oligonucleotide allows thetemperature optima of ligation assays to be expanded.

cDNA Synthesis

The high binding affinity of MGB-oligonucleotide conjugates enableshybridization of shorter oligonucleotides under more stringentconditions. This is important in the synthesis of cDNA from a mRNAtemplate. cDNA synthesis, as commonly practiced, utilizes a reversetranscriptase enzyme to copy a mRNA template into cDNA. The primer forreverse transcription is normally oligodeoxythymidylate, which iscomplementary to the polyadenylate tail found at the 3′ end of most mRNAmolecules. Because hybridization between oligodeoxythymidylate andpolyadenylate is relatively weak, cDNA synthesis reactions must usuallybe conducted under conditions of low stringency. However, under suchconditions, mRNA molecules are known to readily adopt intramolecularsecondary structures, which act as blocks to elongation by reversetranscriptase, leading to production of short, partial cDNA molecules.The increased hybridization strength of MGB-oligonucleotide conjugatesallows cDNA synthesis to proceed under more stringent conditions,wherein secondary structure in the mRNA template is minimized, leadingto the synthesis of longer cDNA products. Hence, a MGB-oligonucleotideconjugate is used as a primer for cDNA synthesis and is extended by apolymerizing enzyme in the synthesis of a cDNA molecule. As an example,oligodeoxythymidylate conjugated to a MGB is used as a primer for cDNAsynthesis. MGB-oligonucleotide conjugates in which the oligonucleotidesequence is complementary to an internal region of a mRNA template, canalso be used for cDNA synthesis. Similarly, MGB-oligonucleotideconjugates ban be used in procedures such as cDNA indexing, describedsupra. Accordingly, use of the methods and compositions of the inventionallows longer cDNA molecules to be obtained, compared to those obtainedby the practices of the prior art.

Nucleic Acid Sequencing Systems

In one embodiment of the invention, a collection of all possible n-meroligonucleotides (where n is an integer less than about 10) are used ina hydrolyzable probe assay to determine a nucleotide sequence. Eacholigonucleotide is uniquely labeled and analysis of released labelindicates which of the oligonucleotides has hybridized to the targetsequence. Alignment of the sequences of the oligonucleotides which havehybridized provides the nucleotide sequence.

MGB-oligonucleotide conjugates are also useful in primer-dependentmethods of DNA sequencing, such as the chain-termination method and itsderivatives, originally described by Sanger et al., supra. Use ofMGB-oligonucleotide conjugates in chain-termination sequencing allowsthe use of shorter primers at higher stringency, and enables a greaterdegree of mismatch discrimination during sequencing. Examples include,but are not limited to, a search for genes sharing a short region ofhomology (on the order of a few amino acids) and sequencing in a regionin which very little existing sequence information is available.MGB-oligonucleotide conjugates are useful in such short primersequencing techniques.

Oligonucleotide Arrays

In another embodiment of the present invention, MGB-oligonucleotideconjugates are also useful in procedures which utilize arrays ofoligonucleotides, such as sequencing by hybridization and array-basedanalysis of gene expression. In these procedures, an ordered array ofoligonucleotides of different known sequences is used as a platform forhybridization to one or more test polynucleotides, nucleic acids ornucleic acid populations. Determination of the oligonucleotides whichare hybridized and alignment of their known sequences allowsreconstruction of the sequence of the test polynucleotide. See, forexample, U.S. Pat. Nos. 5,492,806; 5,525,464; 5,556,752; and PCTPublications WO 92/10588 and WO 96/17957. Materials for construction ofarrays include, but are not limited to, nitrocellulose, glass, siliconwafers, optical fibers and other materials suitable for construction ofarrays such as are known to those of skill in the art.

A major problem with current array-based sequencing and analysis methodsis that, since the different oligonucleotides in an array will havedifferent base compositions, each will have a different T_(m). Hence, itis difficult to determine the stringency conditions that will providemaximum sensitivity, while retaining the ability to distinguishsingle-base mismatches, which is a particularly important considerationfor most, if not all, applications of array technology. Use ofMGB-oligonucleotide conjugates in array-based sequencing and analysistechniques provides a solution to this problem, because conjugation of aMGB to an oligonucleotide makes its T_(m) relatively independent of basecomposition. Thus, for a population of MGB-oligonucleotide conjugates ofa given length, the T_(m) for a perfect hybrid falls within a relativelynarrow temperature range regardless of sequence. At the same time, theT_(m) for a single nucleotide mismatch is well below the T_(m) of theperfect match. See Examples 5 and 6, Tables 11 and 12, supra, where dataare presented to show that, while differences in T_(m) related to basecomposition are minimized for MGB-oligonucleotide conjugates, theconjugates nevertheless retain their discriminatory ability. Thus,arrays designed such that all oligonucleotides are the same length andare present as their MGB conjugates exhibit minimal variation in T_(m)among the different oligonucleotides in the array, enabling more uniformhybridization conditions for the entire array. A further advantage tothe use of MGB-oligonucleotide conjugates in these techniques is that itprovides greater sensitivity, by allowing the use of shorteroligonucleotides, at higher temperatures (and hence higher stringency),while retaining single-nucleotide resolution.

An additional application of the present invention to array technologyis in the examination of patterns of gene expression in a particularcell or tissue. In this case, oligonucleotides or polynucleotidescorresponding to different genes are arrayed on a surface, and a nucleicacid sample from a particular cell or tissue type, for example, isincubated with the array under hybridization conditions. Detection ofthe sites on the array at which hybridization occurs allows one todetermine which oligonucleotides have hybridized, and hence which genesare active in the particular cell or tissue from which the sample wasderived.

Array methods can also be used for identification of mutations, wherewild-type and mutant sequences are placed in an ordered array on asurface. Hybridization of a polynucleotide sample to the array understringent conditions, and determination of which oligonucleotides in thearray hybridize to the polynucleotide, allows determination of whetherthe polynucleotide possesses the wild-type or the mutant sequence. Theincreased discriminatory abilities of MGB-oligonucleotide conjugates areespecially useful in this application of array technology.

Structural Considerations

Oligonucleotide, polynucleotide and nucleic acid are usedinterchangeably to refer to single- or double-stranded polymers of DNAor RNA (or both) including polymers containing modified ornon-naturally-occurring nucleotides, or to any other type of polymercapable of stable base-pairing to DNA or RNA including, but not limitedto, peptide nucleic acids (Nielsen et al. (1991) Science 254:1497-1500;and Demidov et al. (1995) Proc. Natl. Acad. Sci. USA 92:2637-2641),bicyclo DNA oligomers (Bolli et al. (1996) Nucleic Acids Res.24:4660-4667) and related structures. One or more MGB moieties and/orone or more labels, quenching agents, etc. can be attached at the 5′end, the 3′ end or in an internal portion of the oligonucleotide.Preferred MGB moieties include multimers of1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate and multimers ofN-methylpyrrole-4-carbox-2-amide. Particularly preferred are the trimerof 1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate (CDPI₃) and thepentamer of N-methylpyrrole-4-carbox-2-amide (MPC₅).

Modified nucleotides including pyrazolopyrimidines (PCT WO 90/14353; andco-owned U.S. patent application, Attorney Docket No. 34469-20005.00,filed even date herewith), 7-deazapurines and their derivatives arepreferred; additional modified nucleotides are known to those of skillin the art. Oligonucleotides can be obtained from nature or, morepreferably, chemically synthesized using techniques that are well-knownin the art. See, for example, Caruthers, U.S. Pat. No. 4,458,066.Enzymatic synthesis and/or modification of oligonucleotides is alsoencompassed by the present invention. Preferred in the present inventionare DNA oligonucleotides that are single-stranded and have a length of100 nucleotides or less, more preferably 50 nucleotides or less, stillmore preferably 30 nucleotides or less and most preferably 20nucleotides or less.

MGB-oligonucleotide conjugates can comprise one or more modified bases,in addition to the naturally-occurring bases adenine, cytosine, guanine,thymine and uracil. Modified bases are considered to be those thatdiffer from the naturally-occurring bases by addition or deletion of oneor more functional groups, differences in the heterocyclic ringstructure (i.e., substitution of carbon for a heteroatom, or viceversa), and/or attachment of one or more linker arm structures to thebase. All tautomeric forms of naturally-occurring bases, modified basesand base analogues are useful in the MGB-oligonucleotide conjugates ofthe invention.

Similarly, modified sugars or sugar analogues can be present in one ormore of the nucleotide subunits of a MGB-oligonucleotide conjugate.Sugar modifications include, but are not limited to, attachment ofsubstituents to the 2′, 3′ and/or 4′ carbon atom of the sugar, differentepimeric forms of the sugar, differences in the α- or β-configuration ofthe glycosidic bond, anomeric changes, etc. Sugar moieties include, butare not limited to, pentose, deoxypentose, hexose, deoxyhexose, ribose,deoxyribose, glucose, arabinose, pentofuranose, xylose, lyxose, andcyclopentyl.

Modified internucleotide linkages can also be present inMGB-oligonucleotide conjugates. Such modified linkages include, but arenot-limited to, peptide, phosphate, phosphodiester, phosphotriester,alkylphosphate, alkanephosphonate, thiophosphate, phosphorothioate,phosphorodithioate, methylphosphonate, phosphoramidate, substitutedphosphoramidate and the like. Various modifications of bases, sugarsand/or internucleotide linkages, that are compatible with their use inoligonucleotides serving as probes and/or primers, will be apparent tothose of skill in the art.

Modified bases for use in the present invention also include, but arenot limited to, pyrazolo[3,4-d]pyrimidine analogues of adenine andguanine, as disclosed in co-owned PCT Publication WO 90/14353. Preferredbase analogues of this type include the guanine analogue6-amino-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one (ppG) and the adenineanalogue 4-amino-1H-pyrazolo[3,4-d]pyrimidine (ppA). Also of use is thexanthine analogue 1H-pyrazolo[3,4-d]pyrimidin-4(5H)-6(7H)-dione (ppX).These base analogues, when present in an oligonucleotide, strengthenhybridization and improve mismatch discrimination. See co-owned,copending U.S. patent application, Attorney Docket No. 34469-20005.00,filed even date herewith. Example 3, infra, shows the beneficial effectof including a pyrazolo[3,4-d]pyrimidine base analogue in aMGB-oligonucleotide conjugate used in a hydrolyzable probe assay (FIGS.5 and 6). Modified bases for use in the present invention can alsoinclude those for use in selective binding complementaryoligonucleotides, as disclosed in co-owned PCT Publication WO97/12896.Particularly preferred are those which modify the minor groove, such as2-amino adenine and 2-thiothymine.

MGB-oligonucleotide conjugates can contain other pendant moieties, inaddition to the MGB. Examples include, but are not limited to,detectable labels (see elsewhere in the present specification);crosslinking agents such as those disclosed in U.S. Pat. No. 5,659,022and PCT Publications WO 90/14353, WO93/03736, WO94/17092, andWO96/40711; tail moieties such as those disclosed in U.S. Pat. Nos.5,419,966 and 5,512,667; peptide linkers such as those disclosed in U.S.Pat. No. 5,574,142; sterols and other lipohilic groups such as thosedisclosed in U.S. Pat. No. 5,646,126; intercalating agents, reportergroups, electrophilic groups and chelating agents, such as are known tothose of skill in the art, and other pendant moieties known to those ofskill in the art.

The MGB can be attached at either or both ends of the oligonucleotide.In addition or alternatively, one or more MGBs can be attached in theinterior of the oligonucleotide, depending on the length of theoligonucleotide. In general, conjugation of a MGB to either end of anoligonucleotide would provide the greatest degree of hybrid stability,since melting of an oligonucleotide duplex begins at the termini.Nonetheless, if both ends of a duplex formed by an oligonucleotide arerelatively stable, for example, due to a high G+C content, attachment ofa MGB in the interior of an oligonucleotide (for instance, near anA+T-rich sequence) could also enhance stability. The intended use of theMGB-oligonucleotide conjugate may also place limitations on the locationof the conjugated MGB. For instance, if an oligonucleotide is designedto be used as a primer, the 3′-hydroxy group must be free and capable ofbeing elongated by a polymerizing enzyme. Alternatively, an assay thatrequires an oligonucleotide possessing a labeled 5′-end would requireinternal or 3′-end attachment of a MGB.

The location of a MGB within a MGB-oligonucleotide conjugate might alsoaffect the discriminatory properties of such a conjugate. An unpairedregion within a duplex will result in changes in the shape of the minorgroove in the vicinity of the mispaired base(s). Since MGBs fit bestwithin the minor groove of a perfectly-matched DNA duplex, mismatchesresulting in shape changes in the minor groove would reduce bindingstrength of a MGB to a region containing a mismatch. Hence, the abilityof a MGB to stabilize such a hybrid would be decreased, therebyincreasing the ability of a MGB-oligonucleotide conjugate todiscriminate a mismatch from a perfectly-matched duplex. On the otherhand, if a mismatch lies outside of the region complementary to aMGB-oligonucleotide conjugate, discriminatory ability for unconjugatedand MGB-conjugated oligonucleotides of equal length is expected to beapproximately the same. Since the ability of an oligonucleotide probe todiscriminate single base pair mismatches depends on its length, shorteroligonucleotides are more effective in discriminating mismatches. Theprimary advantage of the use of MGB-oligonucleotides conjugates in thiscontext lies in the fact that much shorter oligonucleotides compared tothose used in the prior art (i.e., 20-mers or shorter), having greaterdiscriminatory powers, can be used, due to the pronounced stabilizingeffect of MGB conjugation.

It has also been discovered that substitution of inosine for guanosinein a MGB-oligonucleotide conjugate can enhance hybrid stability. Withoutwishing to be bound by any particular theory, it is likely that inosinesubstitution makes the local shape of the minor groove more favorablefor interaction with a MGB, thereby increasing the strength of theMGB-minor groove interaction. This contribution to duplex stabilityoffsets the weaker base-pairing of the I:C base pair compared to the G:Cbase pair. Example 4 provides data showing increased T_(m)s for hybridsin which one of the strands is an inosine-containing oligonucleotide.

It will be apparent to those of skill in the art that additional minorgroove binding moieties, related to those disclosed herein, willfunction similarly to facilitate hybridization and primer function ofoligonucleotides.

Labels

MGB-oligonucleotide conjugates can be labeled with any label known inthe art of nucleic acid chemistry. Detectable labels or tags suitablefor use with nucleic acid probes are well-known to those of skill in theart and include, but are not limited to, radioactive isotopes,chromophores, fluorophores, chemiluminescent and electrochemiluminescentagents, magnetic labels, immunologic labels, ligands and enzymaticlabels. Suitable labels further include mass labels and those used indeconvolution of combinatorial chemistry libraries, for example, tagsthat can be recognized by high performance liquid chromatography (HPLC),gas chromatography, mass spectrometry, etc.

Methods for probe labeling are well-known to those of skill in the artand include, for example, chemical and enzymatic methods. By way ofexample, methods for incorporation of reactive chemical groups intooligonucleotides, at specific sites, are well-known to those of skill inthe art. Oligonucleotides containing a reactive chemical group, locatedat a specific site, can be combined with a label attached to acomplementary reactive group (e.g., an oligonucleotide containing anucleophilic reactive group can be reacted with a label attached to anelectrophilic reactive group) to couple a label to a probe by chemicaltechniques. Exemplary labels and methods for attachment of a label to anoligonucleotide are described, for example, in U.S. Pat. No. 5,210,015;Kessler (ed.), Nonradioactive Labeling and Detection of Biomolecules,Springer-Verlag, Berlin, 1992; Kricka (ed.) Nonisotopic DNA ProbeTechniques, Academic Press, San Diego, 1992; Howard (ed.) Methods inNonradioactive Detection, Appleton & Lange, Norwalk, 1993. Non-specificchemical labeling of an oligonucleotide can be achieved by combining theoligonucleotide with a chemical that reacts, for example, with aparticular functional group of a nucleotide base, and simultaneously orsubsequently reacting the oligonucleotide with a label. See, forexample, Draper et al. (1980) Biochemistry 19:1774-1781. Enzymaticincorporation of label into an oligonucleotide can be achieved byconducting enzymatic modification or polymerization of anoligonucleotide using labeled precursors, or by enzymatically addinglabel to an already-existing oligonucleotide. See, for example, U.S.Pat. No. 5,449,767. Examples of modifying enzymes include, but are notlimited to, DNA polymerases, reverse transcriptases, RNA polymerases,etc. Examples of enzymes which are able to add label to analready-existing oligonucleotide include, but are not limited to,kinases, terminal transferases, ligases, glycosylases, etc.

In certain embodiments of the present invention, MGB-oligonucleotideconjugates comprising fluorescent labels (fluorophores) and/orfluorescence quenching agents are used. In a preferred embodiment, aMGB-oligonucleotide conjugate contains both a fluorophore and aquenching agent. Fluorescent labels include, but are not limited to,fluoresceins, rhodamines, cyanines, phycoerythrins, and otherfluorophores as are known to those of skill in the art. Quenching agentsare those substances capable of absorbing energy emitted by afluorophore so as to reduce the amount of fluorescence emitted (i.e.,quench the emission of the fluorescent label). Different fluorophoresare quenched by different quenching agents. In general, the spectralproperties of a particular fluorophore/quenching agent pair are suchthat one or more absorption wavelengths of the quencher overlaps one ormore of the emission wavelengths of the fluorophore. A preferredfluorophore/quencher pair is fluorescein/tetramethylrhodamine;additional fluorophore/quencher pair can be selected by those of skillin the art by comparison of emission and excitation wavelengthsaccording to the properties set forth above.

For use in an amplification assay which involves elevated temperatures,such as PCR, or other procedures utilizing thermostable enzymes, thelabel will be stable at elevated temperatures. For assays involvingpolymerization, the label will be such that it does not interfere withthe activity of the polymerizing enzyme. Label can be present at the 5′and/or 3′ end of the oligonucleotide, and/or may also be presentinternally. The label can be attached to any of the base, sugar orphosphate moieties of the oligonucleotide, or to any linking group thatis itself attached to one of these moieties.

EXAMPLES Example 1 MGB-Oligonucleotide Conjugates as PCR Primers

In this example, we show that a modification which greatly improveshybrid stability of a short oligonucleotide also allows theoligonucleotide to serve as a PCR primer. CDPI₃; the trimer of1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate, or CDPI; is asynthetic non-reactive derivative of a subunit of the antitumorantibiotic CC-1065 (Hurley et al. (1984) Science 226:843-844). Thisoligopeptide is a DNA minor groove binder (MGB), with a very highaffinity for the minor groove of A-T-rich double-stranded DNA. It haspreviously been reported that, when compared to unmodifiedoligonucleotides of the same length, CDPI₃-oligonucleotide conjugatesform unusually stable and specific hybrids with complementarysingle-stranded DNA (Lukhtanov et al (1995) Bioconjugate Chem.6:418-426; Afonina et al. (1996) Proc. Nail. Acad. Sci. USA93:3199-3204). This example demonstrates that conjugates of shortoligonucleotides with CDPI₃ make effective primers for PCR, thusimproving the yield and accuracy of priming with short primers.Oligonucleotides as short as 8-mers and G-C-rich 6-mers are able tospecifically prime the amplification reaction when conjugated to a MGB.Thus, conjugation of a MGB to an oligonucleotide under the conditionsdescribed herein does not interfere with the ability of theoligonucleotide 3′-end to be extended by a polymerizing enzyme.

Oligonucleotides and Oligonucleotide Conjugates

Oligonucleotides used in this study were complementary to variousregions of the M13mp19 genome. Oligonucleotide synthesis was performedon an Applied Biosystems Model 394 DNA synthesizer using the 1 μmolcoupling program supplied by the manufacturer. CDPI₃ waspostsynthetically conjugated to the 5′-end of ODNs as described(Lukhtanov et al., supra). ODNs were purified by HPLC on a reverse-phasecolumn eluted by an acetonitrile gradient (usually 0-45%) in 100 mMtriethylamine acetate (pH 7.5) buffer. Purity of unmodified ODNs wasevaluated by electrophoresis on an 8% polyacrylamide-8 M urea gel withsubsequent visualization by silver staining (Daiichi). Purity of theoligonucleotide-CDPI₃ conjugates was verified by analytical HPLC asdescribed above. All oligonucleotide preparations were >95% pure.

The preparation of CDPI₃ is described supra in Reaction scheme 1 andaccompanying text, and formation of the MGB-oligonucleotide conjugatewas accomplished by reaction of the 2,3,5,6-tetrafluorophenyl ester ofCDPI₃ with an oligonucleotide with a 5′-aminohexyl phosphate ester (seeReaction Scheme 3, supra). FIG. 2 depicts the CDPI₃ molecule and thestructures of the linkers through which it is attached to the 5′ or 3′ends of an oligonucleotide. The sequences of the oligonucleotides usedin this example are shown in Table 6.

Thermal Denaturation Studies

Hybrids formed between MGB-oligonucleotide conjugates or unmodifiedoligonucleotides and their complements were melted at a rate of 0.5°C./min in 140 mM KCl, 10 mM MgCl₂ and 20 mM HEPES-HCl (pH 7.2) on aLambda 2S (Perkin Elmer) spectrophotometer with a PTP-6 automaticmulticell temperature programmer. Each oligonucleotide (2 μM) was mixedwith sufficient complementary oligonucleotide to give a 1:1 ratio. Priorto melting, samples were denatured at 100° C. and then cooled to thestarting temperature over a 10 min period. The melting temperatures(T_(m)) of the hybrids were determined from the derivative maxima andcollected in Table 6. In the “sequence” column of Table 6, MGB refers tothe presence of CDPI₃ conjugated through a hexylamine linker esterifiedto the 5′-phosphate group of the oligonucleotide (see FIG. 2).

TABLE 6 Properties of oligonucleotides (ODNs) used in this study Length% Location on SEQ ID ODN T_(m)(° C.) (ntds) GC Sequence M13mp19 genomeNO.  1 45 16 37.5 5′-ATAAAACAGAGGTGAG-3′ complementary to 4937-4922 1  239 12 33.3 5′-ATAAAACAGAGG-3′ complementary to 4937-4926 2  2-C 56 1233.3 5′-MGB-ATAAAACAGAGG-3′ complementary to 4937-4926 2  3 24 10 205′-ATAAAACAGA-3′ complementary to 4937-4928 3  3-C 46 10 205′-MGB-ATAAAACAGA-3′ complementary to 4937-4928 3  4 50 16 43.85′-TAATAACGTTCGGGCA-3′ 4630-4645 4  4-C 66 16 43.85′-MGB-TAATAACGTTCGGGCA-3′ 4630-4645 4  5 16 6 33.3 5′-ATAACG-3′4632-4637 5  5-C 36 6 33.3 5′-MGB-ATAACG-3′ 4632-4637 5  6-C 57 12 33.35′-MGB-TAATAACGTTCG-3′ 4630-4641 6  7-C 49 10 20 5′-MGB-TAATAACGTT-3′4630-4639 7  8 25 8 62.5 5′-CGGGCAAA-3′ 4640-4647 8  8-C 33 8 62.55′-MGB-CGGGCAAA-3′ 4640-4647 8  9 47 16 43.8 5′-CGGGCAAAGGATTTAA-3′4640-4655 9  9-C 53 16 43.8 5′-MGB-CGGGCAAAGGATTTAA-3′ 4640-4655 9 10<17 6 50 5′-GGCAAA-3′ 4642-4647 10 10-C <17 6 50 5′-MGB-GGCAAA-3′4642-4647 10 11 23 8 62.5 5′-CGGCTCTA-3′ 4720-4727 11 11-C 37 8 62.55′-MGB-CGGCTCTA-3′ 4720-4727 11 12 43 16 37.5 5′-CGGCTCTAATCTATTA-3′4720-4735 12 12-C 52 16 37.5 5′-MGB-CGGCTCTAATCTATTA-3′ 4720-4735 12 1334 16 18.75 5′-TATTTTAGATAACCTT-3′ 4756-4771 13 13-C 56 16 18.755′-MGB-TATTTTAGATAACCTT-3′ 4756-4771 13

PCR Reactions

All PCR reactions were performed on a Perkin-Elmer Cetus DNAThermocycler and included: PCR buffer (Promega) with no magnesium, 1.6μmM MgCl₂, 50 μM dNTP, 50 nM each primer, 0.2 μg M13mp19 DNA and 1-2Units Taq DNA polymerase (Promega). Final volume for each reaction was50 μl. The standard PCR profile was as follows: 3 min at 94° C., 30cycles of 1 min at 94° C., 1.5 min at annealing temperature and 30 s at72° C., finally followed by 5 min at 72° C. and a 4° C. soak. For the8-mers and 6-mers, PCR was performed in the touch-down manner (Don, R.H. et al. (1991) Nucleic Acids Res. 19: 4008) with a starting annealingtemperature of 55° C. for 8-mer primers and 50° C. for 6-mers. Eachsubsequent cycle had an annealing temperature 1° C. lower until 41° C.(for 8-mers) or 37° C. (for 6-mers) was reached, with the final 15cycles annealed at these final temperatures. Touch-down PCR has beenshown to maximize the yield of product when using short primers (Don, R.H. et al., supra). Amplifications with 16-mer, 12-mer and 10-mer primerswere analyzed by electrophoresis of 10 μl of the reaction mixture on a2% agarose gel and detection of the bands by ethidium bromide staining.Amplifications with 8-mer and 6-mer primers were analyzed byelectrophoresis of 5 μl of reaction mixture on an 8% polyacrylamidesequencing gel and detection of the bands by silver staining (Daiichi).

Results

Hybrid Stability

Table 6 presents the melting temperatures (T_(m)s) of duplexes formed bythe MGB-ODN conjugates with complementary ODNs, showing the effect of aterminally-conjugated CDPI₃ group on duplex stability for duplexes ofdifferent lengths and G-C compositions. For the 16-mer duplexes, thelargest increase in T_(m) attributable to a tethered MGB (22° C.), wasobtained when that group was flanked by a run of seven A-T base pairs(compare oligonucleotides 13-C and 13). A-T-rich sequences of thislength form minor grooves which act as good binding sites for the CDPI₃tripeptide. Of all the hybrids examined, the 16-mer duplex formed byoligonucleotide 4-C gave the highest absolute T_(m) (66° C.). Thisunusually high T_(m) reflects an otherwise G-C-rich duplex whichcontains six A-T base pairs adjacent to the tethered CDPI₃ group.Conversely, a 16-mer duplex with a O—C-rich sequence flanking the MGBconjugation site (12-C) was only 9° C. more stable than the unmodifiedduplex (12). The CDPI₃ group in this duplex binds in a less favorableG-C-rich minor groove.

The T_(m)s reported for the shorter primers in Table 6 follow the sametrends as for the 16-mers. The primers with the A-T-rich regionsadjacent to the MGB at the 5′-end had higher T_(m)s than those withG-C-rich regions, and they showed a greater increase in T_(m) comparedto their non-conjugated counterparts. The 10-mers 3-C and 7-C, forinstance, had T_(m) values of 46-49° C., well within a range adequatefor specific PCR priming.

Priming Ability

The ODNs and MGB-ODN conjugates were tested as PCR primers using M13mp19single-stranded DNA as the amplification substrate. Typically,unmodified and CDPI₃-conjugated versions of the same oligonucleotidewere compared. These were tested in parallel, as reverse primers, usinga PCR profile in which only the annealing temperature was varied. Alower than usual concentration of primers (<0.1 μM) was employed whenusing the MGB-oligonucleotide conjugates. This minimized any spuriousinteraction of these conjugates with A-T-rich sequences due to theanchor effect of the CDPI₃ group (Afonina, I. et al., supra). To confirmthe specificity of primer binding, the primers were designed such thatevery amplified product it this study contained a DdeI restriction site.In addition to measuring the size of the amplification product (FIGS. 3and 4), aliquots of selected PCR reaction mixtures were treated withDdeI and analyzed in a 2% agarose gel. In all cases the expectedrestriction fragments were obtained.

FIGS. 3A and 3B demonstrate the improved priming performance of 16-merMGB-ODN conjugates in comparison with unmodified primers. Conditions ofamplification were the same with the exception of annealing temperature,which was 45° C. for FIGS. 3A and 68° C. for FIG. 3B. While all of these16-mer oligonucleotides primed at the lower temperature (FIG. 3A, seelegend for predicted product sizes), only those with a 5′-CDPI₃ groupprimed at the higher temperature (FIG. 3B, see legend for predictedproduct sizes).

FIG. 3C confirms the advantages of conjugation of CDPI₃ to short primers10 or 12 nucleotides long. Both conjugated primers efficiently amplifiedthe expected sequence (see FIG. 3C legend for predicted product sizes).The same primers without a tethered MGB did not generate productdetectable by ethidium bromide staining when amplified under the sameconditions.

FIG. 4 demonstrates that specific priming is possible even for primersas short as an 8-mer (FIG. 4A) and a 6-mer (FIG. 4B). A 10-mer forwardprimer was used in these reactions. The low levels of productnecessitated the use of touch-down PCR (Don, et al., supra) anddetection of bands by silver staining. In each case a band of expectedsize (see legends to FIGS. 4A and 4B for predicted product sizes) wasobserved only when the reverse primer was conjugated to a CDPI₃ group.

Without wishing to be bound by any particular theory, it appears thatthe increase in stability of a hybrid comprising a MGB-oligonucleotideconjugate compared to one containing a non-conjugated oligonucleotide islikely to be due to the binding of the tethered MGB in the duplexregion. The binding region of the MGB probably spans up to 6 base pairs.Importantly, the 3′-terminus of the MGB-conjugated oligonucleotide isstill recognized by the polymerizing enzyme, as primer extension seemsto depend only on hybrid stability and is not inhibited by the presenceof the MGB.

Example 2 Use of MGB-Oligonucleotide Conjugates in a Hydrolyzable ProbeAssay

In this example, we show that conjugation of MGBs to shortoligonucleotides results in improved hybrid stability and improveddiscrimination between a perfect hybrid and a single-base mismatch, whenMGB-short oligonucleotide conjugates are used in a hydrolyzable probeassay. The procedure described by Wittwer et al. (1997a) BioTechniques22:130-138, was used. In this method, MGB-oligonucleotide conjugates,additionally comprising a fluorophore and a quenching agent, were usedas probes in a hydrolyzable probe assay. This type of probe is designedto be complementary to a predicted amplification product, and emits verylittle or no fluorescence, due to the proximity of the fluorophore tothe quenching agent. Formation of a hybrid between the probe and theamplification product produces a structure that is a substrate forexonucleolytic hydrolysis of the probe by a polymerase possessingduplex-specific exonuclease activity, if the polymerase has initiatedpolymerization at an upstream primer. The exonuclease action willrelease the fluorophore from the hybridized oligonucleotide and hencefrom the proximity of the quenching agent, resulting in an increase influorescence. Thus, in this assay, increase in fluorescence is dependentupon duplex formation between the fluorophore/quencher-labeledMGB-oligonucleotide conjugate probe and the desired amplificationproduct. See U.S. Pat. No. 5,210,015; Livak et al. (1995) PCR Meth. App.4:357-362; and Heid et al. (1996) Genome Res. 6:986-994 for furtherdetails.

Synthesis of MGB-Oligonucleotide Conjugates Containing Fluorophore(s)and Quencher(s)

CDPI₃-CPG Supports (Scheme 6)

4-[(2-Phenyl)-1,3-dioxolan-4-yl]-1-butanol (17). To a solution of1,2,6-trihydroxyhexane (10.0 g, 74.6 mmol) and benzaldehydedimethylacetal (15.0 g, 98.7 mmol) in dry DMF (10 mL) was addedAmberlyst 15 (5.0 g). The mixture was stirred at 100° C. for 5 min, thencooled and filtered. The filtrate was concentrated and residue obtainedwas re-dissolved in ethyl acetate. After being washed with water andbrine, the solution was dried over Na₂SO₄. The crude product obtainedafter evaporation of the solvent was chromatographed on silica elutingwith ethyl acetate. Concentration of the proper fractions afforded 11.0g (66%) of the title product (a mixture of diastereomers) as a colorlessoil: ¹H NMR (CDCl₃, 300 MHz, ppm) 7.48 (m, 2H), 7.39 (m, 3H), 5.92 (s,0.4H), 5.80 (s, 0.6H), 4.3-4.1 (m, 2H), 3.64 (m, 3H), 1.8-1.4 (m, 6H).

2-Phenyl-4-(4-phtalimidobut-1-yl)-1,3-dioxolane (18). A solution ofdiethyl azodicarboxylate (8.6 g, 49.4 mmol) in 40 mL of THF was slowlyadded to a cold (ice bath) solution of 17 (10.0 g, 45.0 mmol),triphenylphosphine (13.0 g, 49.5 mmol) and phthalimide (7.3 g, 49.7mmol) in 60 mL of THF. After being kept at ambient temperatureovernight, the solution was concentrated and the residue was trituratedwith ether (˜150 mL) to precipitate triphenylphosphine oxide which wasthen removed by filtration. The filtrate was concentrated to give anoily residue which was chromatographed on silica eluting with 33% ethylacetate in hexane. Concentration of the pure product fiactions followedby drying under vacuum afforded 14.6 g (92%) of the desired product as asemi-solid (a mixture of diastereomers): ¹H NMR (CDCl₃, 300 MHz, ppm)7.85 (m, 2H), 7.71 (m, 2H), 7.46 (m, 2H), 7.36 (m, 3H), 5.90 (s, 0.4H),5.78 (s, 0.6H), 4.3-4.0 (m, 2H), 3.68 (m, 3H), 1.9-1.3 (m, 6H).

6-[N-(9-Fluorenylmethoxycarbonyl)amino]-(R,S)-1,2-hexanediol (19). Asuspension of 18 (13.5 g, 38.5 mmol) in 50 mL of ethanol was treatedwith 1.9 mL (39.1 mmol) of hydrazine monohydrate and heated at refluxfor 3 h. The mixture was cooled and acidified with 1M HCl to pH 3. Theprecipitate formed was filtered off, the filtrate was extracted withether and concentrated to give a semi-solid. It was suspended in2-propanol to separate insoluble inorganic salts. After filtration, thesolution was concentrated in vacuo to afford 5.0 g of a syrup containingmostly the desired product contaminated with 2-propanol. This materialwas used in the next reaction without additional purification.

To a solution of the above crude aminodiol in a mixture of methanol (25mL) and CH₂Cl₂ (10 mL) was added triethylamine (7 mL) followed by9-fluorenylmethyl N-succinimidyl carbonate (8.0 g, 23.7 mmol). Afterbeing stirred for 1 h, the reaction mixture was treated with acetic acid(5 mL) to neutralize excess triethylamine, and concentrated. Triturationof the residue with water gave a white solid which was washed with waterand ether. Drying in vacuo afforded 5.5 g (40%) of analytically pureproduct: ¹H NMR (DMSO-d6, 300 MHz, ppm) 7.88 (d, 2H), 7.68 (d, 2H), 7.41(t, 2H), 7.33 (it 2H), 4.45 (t, 1H), 4.37 (d, 1H), 4.28 (d, 2H), 4.21(m, 1H), 3.35 (m, 1H), 3.24 (m, 2H), 2.96 (m, 2H), 1.39 (m, 4H), 1.21(m, 2H).

6-[N-(9-Fluorenylmethoxycarbonyl)amino]-2-O-(4,4′-dimethoxytriphenylmethyl)-(R,S)-1,2-hexanediol(20). To a solution of 19 (2.0 g, 5.6 mmol) in 20 mL of dry pyridine wasadded N,N-dimethylaminopyridine (0.1 g) and 4,4′-dimethoxytritylchloride (4.0 g, 11.8 mmol). After being stirred for 2 h, the solutionwas concentrated and the resultant oily residue was re-dissolved inethyl acetate. The solution was washed with water, brine and dried overNa₂SO₄. Evaporation of the solvent gave crude product which waschromatographed on silica eluting with 50% ethyl acetate in hexane. Thetitle product was obtained as a pale yellow amorphous solid (2.8 g, 79%)after evaporation of the solvent: (CDCl₃, 300 MHz, ppm) 7.77 (d, 2H),7.59 (d, 2H), 7.5-7.1 (m, 17H), 6.84 (t, 4H), 4.80 (t, 1H), 4.40 (d,2H), 4.22 (t, 1H), 3.80 (s, 6H), 3.2-3.0 (m, 4H), 1.6-1.1 (m, 6H).

2,3,5,6-Tetrafluorophenyl-6-[N-(9-fluorenylmethoxycarbonyl)amino]-(R,S)-2-(4,4′-dimethoxytriphenylmethoxy)-hex-1-ylbutanedioate (21). To a solution of 20 (1.0 g, 1.6 mmol) in 5 mL of drypyridine was added succinic anhydride (1.0 g, 10 mmol) followed by1-methylimidazole (0.02 mL). The reaction mixture was stirred for 8 h at55° C. and treated with water (1 mL). Concentration under vacuum gave anoil which was partitioned between CHCl₃ and cold 10% citric acid. Theorganic phase was washed with water and dried over Na₂SO₄. Evaporationof the solvent afforded crude acid as an amorphous solid (0.99 g), thismaterial was taken to the next step without further purification.

To a solution of the above acid in 5 mL of dry CH₂Cl₂ was addedtriethylamine (0.25 mL) followed by 0.25 mL (1.4 mmol)2,3,5,6-tetrafluorophenyl trifluoroacetate. Gamper, H. B. et al. (1993)Nucleic Acids Res. 21: 145. After being kept at ambient temperature for15 min, the solution was applied onto a silica gel column. Elution ofthe column with 33% ethyl acetate in hexane and concentration of thepure product fractions afforded 1.0 g (71%) of the desired TFP ester asa white, amorphous solid: (CDCl₃, 300 MHz, ppm) 7.76 (d, 2H), 7.58 (d,2H), 7.5-7.1 (m, 17H), 7.00 (m, 1H), 6.84 (t, 4H), 5.1 (m, 1H), 4.79 (t,1H), 4.38 (d, 2H), 4.22 (t, 1H), 3.78 (s, 6H), 3.2-2.9 (m, 6H), 2.81 (m,2H), 1.61 (m, 2H), 1.45 (m, 2H), 1.26 (m, 2H).

Preparation of CPG 22. To a solution of 21 (0.5 g, 0.57 mmol) in 20 mLof dry pyridine was added long chain aminoalkyl CPG (500 A) (5.0 g)followed by 1-methylimidazole (1.0 mL). After being swirled for 15 h atambient temperature, the suspension was treated with acetic anhydride (3mL) to cap unreacted amino groups (15 min). The CPG was then washed withDMF, acetone, ether and dried. The CPG was analyzed for DMTr content(Atkinson, T. et al. (1984) Solid-Phase Synthesis ofOligodeoxyribonucleotides by Phosphite-Triester Method. In“Oligonucleotide Synthesis, A practical Approach”. (M. J. Gait, Ed.) pp.35-81. IRL Press, Washington, D.C.) and found to have a loading of 49μmol/g.

Preparation of CPG 23. CPG 22 (2.8 g) was deprotected by a treatmentwith 20 mL of 0.2 M 1,8-diazobicyclo[5.4.0]undec-7-ene(1, 5-5) (DBU) for20 min. The CPG was extensively washed with DMF and ether, dried andre-suspended in a solution of CDPI₃-TFP (Lukhtanov, E. A. et al. (1995)Bioconjugate Chem., 6: 418) (200 mg) and N,N-diisopropylethylamine (1mL) in 8 mL of DMF. After being swirled for 3 days, the CPG was washedwith N,N,-dimethylacetamide, acetone, ether and dried. Unreacted aminogroups were capped by treatment with 10% acetic anhydride in pyridinefor 15 min, and the CPG was washed and dried as described above.

Oligonucleotide synthesis using the CDPI₃-CPG support (Scheme 7).Trityl-off 3′-CDPI₃ oligonucleotides were prepared in 1 μmol scale usingstandard 3′-phosphoramidite chemistry on the CDPI₃-CPG support (˜20-50mg) on an ABI 394 according to the protocol supplied by the manufacturerwith one exception: 0.01 M (instead of the standard 0.1 M) iodinesolution in was utilized in the oxidation step to avoid iodination ofthe CDPI₃ moiety. In order to introduce an amino-linker for thepostsynthetic incorporation of the TAMRA dye (see below), protectedaminopropyl ppG and aminopropyl ppA phosphoramidites were utilized atthe desired step instead of the standard guanosine or adenosinephosphoramidites, respectively. See co-owned, PCT WO 90/14353.

Incorporation of 6-FAM and TET Fluorophores (Scheme 8) and Isolation ofthe Conjugates.

6-FAM (6-carboxyfluorescein) or TET(6-carboxy-4,7,2′,7′-tetrachlorofluorescein) was introduced at the laststep of the above-described automated oligonucleotide synthesis usingcorresponding 6-FAM and TET phosphoramidites (Glen Research) accordingto the protocol supplied by the manufacturer. After deprotection themodified ODNs were purified by reverse-phase chromatography on a 4.6×250mm, C-18 column (Dynamax-300, Rainin) eluting with a gradient ofacetonitrile (0-60%) buffered at pH 7.5 (0.1 M triethylammoniumacetate). The desired fraction was concentrated to a volume of ˜50 μL byextraction with n-butanol and then diluted with 2% solution of LiClO₄ inacetone (1.2 mL). The resultant precipitate was collected bycentrifugation, the pellet was washed with acetone (2×1.2 mL) and driedunder vacuum.

Postsynthesis introduction of TAMRA residue (Scheme 9). TAMRA(tetramethylrhodamine) was incorporated into the above conjugates byreaction of the oligonucleotide with TAMRA-N-hydroxysuccinimide ester(Glen Research) according to the protocol supplied by the manufacturer.Under these conditions, the TAMRA moiety is added to an amino grouplinked to a ppG or ppA residue in the oligonucleotide. Purification ofthe double dye (fluorescein and TAMRA)-labeled CDPI₃-ODN conjugates wasaccomplished by denaturing 20% PAGE, the desired band was cut out andthe conjugate extracted by incubation of the gel slice in 0.1 Mtriethylammonium acetate (10 mL) (pH 7.5) overnight at 37° C. Finally,the conjugates were isolated from the extract by reverse phase HPLC asdescribed above.

Assay

Reaction mixtures contained 40 mM NaCl, 20 mM Tris-Cl pH 8.9, 5 mMMgSO₄, 0.05% (w/v) bovine serum albumin, 125 μM each of the four dNTPs,1 ng template 0.5 μM each primer, 0.5 μM probe (fluorophore/quencherlabeled MGB-oligonucleotide conjugate) and 0.5 μl of Taq Polymerase per10 μl reaction volume. Forty cycles of amplification were conducted.Each cycle was 0 sec at 94° C. for denaturation (i.e., temperature wasraised to 94° C. and immediately lowered to the annealing/elongationtemperature), followed by 15 sec at a combined annealing/elongationtemperature. The annealing/elongation temperature varied among differentreactions and is specified for each particular case.

Assays were conducted following the method of Wittwer et al. (1997a)BioTechniques 22:130-138, and fluorescence measurements were made usinga Light Cycler™, available from Idaho Technology. Wittwer et al. (1997b)BioTechniques 22:176-181. Fluorescein fluorescence was determined byexcitation at 485 nm and detection of emission between 518-530 nm.

Results

Experiments using different length probes, either unconjugated orconjugated to a MGB (CDPI₃) were conducted at variousannealing/elongation temperatures. All probes contained a molecule ofcarboxamidofluorscein conjugated to the 5′-end via a hexyl linker and amolecule of tetramethylrhodamine (TAMRA) conjugated at the 3′-end, asdescribed supra.

The effect of MGB conjugation on hybridization between perfectly-matchedsequences and sequences containing a single nucleotide mismatch wasassessed for 12-mer oligonucleotides in a hydrolyzable probe assay. Ineach experiment, there were four samples, each containing a differentprobe. The probes were either fully complementary to the target sequence(i.e., a perfect match) or had a single base mismatch, and eithercontained or lacked a MGB conjugated at the 3′-end of theoligonucleotide as described infra. The experiment was conducted at anannealing/elongation temperature of 65° C. Oligonucleotides which didnot contain a MGB gave baseline levels of fluorescence through 30 cyclesof amplification. A MGB-conjugated 12-mer with perfect complementarityto target showed gradually increasing fluorescence from the start of theamplification process, with a significant increase in fluorescencebeginning at about the 18th cycle. A MGB-conjugated 12-mer with asingle-nucleotide mismatch showed fluorescence levels that were close tobaseline, and that were clearly distinguishable from the levelsgenerated by the perfectly matched sequence.

The behavior of 10-mer oligonucleotides with and without a conjugatedMGB was also examined at an annealing/elongation temperature of 65° C.With 10-mers, the background was higher and more variable. Nevertheless,while only background signal was obtained when an unconjugated 10-merwas used in the assay (with either a perfect match or asingle-nucleotide mismatch), signal obtained using a MGB-conjugated10-mer with perfect complementarity was readily distinguished from thatobtained using a MGB-conjugated 10-mer with a single-base mismatch.

From these results it is clear that conjugation of a MGB to a shortoligonucleotide greatly stabilizes the hybrids formed by such conjugatedoligonucleotides, compared to oligonucleotides not containing anattached MGB. It should also be noted that, in all cases, the differencein hybrid stability (as evidenced by differences in fluorescence levelsat later amplification cycles) between fully complementary probes andprobes with a single base mismatch is more pronounced for probes with aconjugated MGB (compared to unconjugated probes), showing thatconjugation of a MGB helps to increase the discriminatory power of ashort oligonucleotide probe.

Example 3 Effect of Nucleotide Analogues on Hybridization Strength andDiscriminatory Ability of MGB-Oligonucleotide Conjugates

Further increases in discriminatory ability of a MGB-oligonucleotideconjugate are obtained when the conjugate also contains apyrazolo[3,4-d]pyrimidine nucleotide analogue. In this system, thetarget sequence is located in the E. coli supF gene contained in theplasmid pSP189 (FIG. 5, SEQ ID No.: 40). See Parris et al. (1992) Gene117:1-5. Binding sites for the primers used for amplification areindicated as Primer 1 and Primer 2, with Primer 1 having a sequence andpolarity that is identical to that shown in FIG. 5, and Primer 2 havinga sequence and polarity that is the reverse complement to that shown inFIG. 5. A 15-mer probe, labeled with fluorescein at the 5′-end, and withTAMRA and CDPI₃ at the 3′-end, was designed to be complementary to aregion within the approximately 375 nucleotides between the primers, asindicated in FIG. 5. This probe was tested using a series of templates,each containing a different single-nucleotide mismatch with the probesequence, as shown in FIG. 5 and described infra.

Primer sequences The forward amplification primer has the sequence:5′-CTGGGTGAGCAAAAACAGGAAGGC-3′ SEQ ID No.: 14 The reverse primer has thesequence: 5′-TGTGATGCTCGTCAGGGGGG-3′ SEQ ID No.: 15 Sequence of probe:The 15-mer probe has the following sequence: 5′-GGGTTCCCGAGCGGC SEQ IDNO.: 16

Template Sequences:

The 15-nucleotide region of the template that is complementary to theprobe used in this study was modified to generate a series of pointmutations, as shown in FIG. 5. Each of the mutant templates was used ina separate assay with the 15-mer probe. The mutant sequences within thisregion of the template were as follows, with the mismatched nucleotideindicated by bold underling:

5′-GGGTTCCCGAGCGGC (perfect match) SEQ ID NO.: 17 5′-G A GTTCCCGAGCGGC(32 G-A mismatch) SEQ ID NO.: 18 5′-GGGTT T CCGAGCGGC (36 C-T mismatch)SEQ ID NO.: 19 5′-GGGTT G CCGAGCGGC (36 C-G mismatch) SEQ ID NO.: 205′-GGGTT A CCGAGCGGC (36 C-A mismatch) SEQ ID NO.: 21 5′-GGGTTC TCGAGCGGC (37 C-T mismatch) SEQ ID NO.: 22 5′-GGGTTC A CGAGCGGC (37 C-Amismatch) SEQ ID NO.: 23 5′-GGGTTCCC C AGCGGC (39 G-C mismatch) SEQ IDNO.: 24 5′-GGGTTCCCG T GCGGC (40 A-T mismatch) SEQ ID NO.: 255′-GGGTTCCCGA A CGGC (41 G-A mismatch) SEQ ID NO.: 26 5′-GGGTTCCCGA CCGGC (41 G-C mismatch) SEQ ID NO.: 27 5′-GGGTTCCCGAGC A GC (43 G-Amismatch) SEQ ID NO.: 28 5′-GGGTTCCCGAGC T GC (43 G-T mismatch) SEQ IDNO.: 29 5′-GGGTTCCCGAGCG T C (44 G-T mismatch) SEQ ID NO.: 30

The assay was conducted according to Wittwer (1997a,b, supra). FIG. 6shows that, when a MGB-conjugated 15-mer, additionally having allguanine residues replaced by the guanine analogue6-amino-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one (ppG), is used in an assayin which annealing/elongation temperature is conducted at 75° C.,generation of signal by probes with a single-base mismatch is completelysuppressed, with no effect on the level of signal generated by theperfectly-matched probe.

Thus, the combination of MGB conjugation, substitution with modifiednucleotides and appropriate reaction conditions enable facilediscrimination between a perfect-matched hybrid and a hybrid containinga single-nucleotide mismatch, at high stringency, allowing a heretoforeunparalleled degree of specificity to be obtained in hybridizationreactions with short oligonucleotides.

Example 4 Effects of Base Composition and Oligonucleotide Backbone onHybrid Stability of MGB-Oligonucleotide Conjugates Materials and Methods

Synthesis of Oligonucleotides (ODNs)

All ODNs were prepared from 1 μmol appropriate CPG support on an ABI 394synthesizer using the protocol supplied by the manufacturer. Protectedβ-cyanoethyl phosphoramidites of 2′-deoxyribo and2′-O-methylribonucleotides, CPG supports, deblocking solutions, capregents, oxidizing solutions and tetrazole solutions were purchased fromGlen Research. 5′-Aminohexyl modifications were introduced using anN-(4-monomethoxytrityl)-6-amino-1-hexanol phosphoramidite linker (GlenResearch). 3′-Aminohexyl and 3′-hexanol modifications were introducedusing the CPG prepared as previously described. Petrie et al. (1992)Bioconjugate Chem. 3:85-87. All other general methods employed forpreparative HPLC purification, detritylation and butanol precipitationwere carried out as described. Reed et al. (1991) Bioconjugate Chem.2:217-225. All purified octanucleotides were analyzed by C-18 HPLC(column 250×4.6 mm) in a gradient of 0-30% acetonitrile in 0.1 Mtriethylamine acetate buffer, pH 7.0, over 20 min at a flow rate of 2ml/min. Pump control and data processing were performed using a RaininDynamax chromatographic software package on a Macintosh computer. ODNpurity was further confirmed by capillary gel electrophoresis (CGE) witha P/ACE™ 2000 Series equipped with an eCAP™ cartridge (Beckman,Fullerton, Calif.). The octanucleotides were >95% pure by C-18 HPLC andshowed one moor peak on CGE. Thermal denaturation studies were performedas described. Lukhtanov et al. (1995) Bioconjugate Chem. 6:418-426;Lukhtanov et al. (1996) Bioconjugate Chem. 7:564-567. The meltingtemperatures (T_(max) values) of the hybrids were determined from thefirst derivative maxima (change in A₂₆₀ with respect to time) and areshown in Tables 7-10.

Synthesis of CDPI₃-Tailed ODN Conjugates

Methods for conjugation of the1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate trimer (CDPI₃) toODNs have been published. Lukhtanov et al. (1995), supra. All(CDPI₃)-tailed octanucleotides were isolated from reaction mixtures and,if necessary, repurified on an analytical (4.6×250 mm) PLRP-S column(Polymer Labs) using a gradient of acetonitrile (0-60% for mono- and0-80% for bis-CDPI₃-tailed ODNs) in 0.1 M triethylammonium acetate, pH7.5. The column was incubated at 70-75° C. All ODN-CDPI₃ conjugatesprepared were analyzed and characterized as described. Lukhtanov et al.(1995), supra.

Molar Extinction Coefficients of Octanucleotides and their Derivatives

The concentrations of the octanucleotides and their derivatives weremeasured spectrophotometrically. The molar extinction coefficients(ε₂₆₀) of unmodified octadeoxyribonucleotides were determined bymeasuring the absorption of the ODNs before and after completehydrolysis by snake venom nuclease. Shabarova et al. (1981) NucleicAcids Res. 9:5747-5761. With this value, ³²P-labeled CDPI₃-tailedconjugates with known specific activities were prepared and their 8260values determined as described. Lokhov et al. (1992) Bioconjugate Chem.3:414-419. Molar extinction coefficients of all octadeoxyribonucleotidesand their CDPI₃ derivatives used in this study are, in order ofunmodified ODN, mono-CDPI₃ and di-CDPI₃ derivative: d(pT)₈p, 65.8,110.1, 178.1/mM/cm; d(pA)₈p; 81.9, 150.0, 218.0/mM/cm;d(pApGpCppGpApTpGp), 74.0, 162.9, 230.9/mM/cm; d(pCpApTpCpCpGpCpTp),65.0, 136.9, 204.9/mM/cm. These extinction coefficients were used todetermine the concentration of all other backbone-modifiedoctanucleotides and their CDPI₃ conjugates. To calculate 6260 values fordeoxyinosine-containing ODNs the value of 4.6/mM/cm multiplied by thenumber of hypoxanthine bases was subtracted from the extinctioncoefficients of the corresponding dG-containing ODNs.

Results

CDPI₃ Residue Conjugated to AT-Rich Duplexes

The antibiotic CC-1065 (Reynolds et al. (1985) Biochemistry24:6228-6237) and its numerous synthetic derivatives (Boger et al.(1995) Proc. Nail Acad. Sci. USA 92:3642-3649), including CDPI₃, have astrong affinity for AT-rich sites of double-stranded DNA, as do most ofthe MGBs. CC-1065 also binds to and alkylates AT-rich sequences inRNA-DNA hybrids. Kim et al. (1995) Antisense Res. Dev. 5: 149-154. Weexpected the binding properties of ODN-conjugated CDPI₃ to be similar tothat observed for free CDPI₃. We reported earlier on binding ofoligothymidylates carrying a CDPI_(n) moiety to polyadenylic acids.Lukhtanov et al. (1995, 1996) supra. Here we use the short d(pT)₈.(pA)₈duplex as a model system for a more comprehensive investigation. NMRanalysis of a CC-1065-DNA complex (Scahill et al. (1990) Biochemistry29:2852-2860) and noncompetitive binding of CDPI₃ and ethidium bromideto AT-rich DNA duplexes (roger et al. (1992) J. Org. Chem. 57:1277-1284)have shown that these compounds span 5-6 base pairs in the DNA minorgroove. An octamer duplex, therefore, is a length of double-stranded DNAsufficient to accommodate the conjugated CDPI₃ residue and the linkersused in the present study.

A variety of octa-adenylate and octathymidylate derivatives with DNA,2′-O-methyl RNA or DNA phosphorothioate backbones and carrying CDPI₃residues at either or both termini were prepared. The structures ofCDPI₃ and linkers for 5′- and 3′-tailed ODN conjugates are shown in FIG.2. Complementary duplexes constructed from these sequences were meltedand the T_(max) data are shown in Table 7. Although the complex strandswere taken in equimolar ratio and only single melting transitions wereobserved in all cases, the possibility of higher order structureformation, other than duplex, cannot be completely excluded. However, aprevious study conducted on d(pT)₈.poly(dA)/poly(rA) did not reveal atendency of CDPI₃-tailed ODNs to form triplex structures. Lukhtanov etal. (1995) supra.

As expected, the most dramatic stabilization was achieved for ATduplexes with a regular DNA backbone in both strands. The T_(max) of theweak d(pT)₈.d(pA)₈ complex, 12-14° C. under these conditions, wasincreased to 53-61° C. after one of the strands was conjugated to a MGB.Positioning of the MGB on the duplex gave some strand-specific effects.Location of the CDPI₃ moiety on either the 3′- or 5′-end ofoctathymidylate did not significantly affect duplex stability(T_(max)=56 or 58° C.), but did so when conjugated to oligoadenylatesequences. Octa-adenylate carrying the 3′-CDPI₃ residue formed the moststable complementary complex (T_(max)=61° C.) and the 5′-tailedconjugate the least in this series (T_(max)=53° C.).

Table 8 shows two examples of longer DNA duplexes with terminal AT-richsequences, which were also stabilized by tethered CDPI₃ residues. Thesetetradecanucleotides were designed to test the effect of CDPI₃ bindingin a region of mixed or alternating AT sequences, as opposed to the A₈T₈homopolymeric sequence above. Conjugation of CDPI₃ to the 3′-end ofthese ODNs increased T_(max) values of the complementary duplexes by21-22° C. Although this value is half that observed for d(pT)₈.d(pA)₈duplexes (40-49° C.), the overall free energy contribution of the CDPI₃residue was estimated and found to be comparable in both cases. Thedecrease in ΔT_(max) was expected, since unmodified hexadecanucleotideduplexes (Table 8) were significantly more stable than d(pT)₈.d(pA)₈(T_(max)=48-49 versus 12-14° C.).

Enhancement of nuclease resistance of ODNs by replacement of thephosphodiester group with a phosphorothioate is well established.Eckstein et al. (1970) Eur. J. Biochem. 13:558-564; Agrawal et al.(1991) Proc. Natl. Acad. Sci. USA 88:7595-7599. This modificationgenerally reduces, however, the affinity of the ODN for a complementarysingle-stranded target See, for example, Suggs et al. (1985) NucleicAcids Res. 13:5707-5716; Cosstick et al. (1985) Biochemistry24:3630-3638; LaPlanche et al. (1986) Nucleic Acids Res. 14:9081-9093;and Stein et al. (1988) Nucleic Acids Res. 16:3209-3221. As we foundhere, d(PT)₈.d(pA)₈ duplexes having a phosphorothioate backbone ineither the d(pT)₈ strand or both strands were significantly destabilizedand showed no melting transition over 0° C. (Table 7). Thephosphorothioate analog of d(pA)₈ formed a weak hybrid with unmodifiedd)₈ (T_(max)=8-9° C.). Conjugation of a single CDPI₃ residue increasedthe T_(max) of all these duplexes into a melting range of 35-56° C., astabilization effect in some cases of >45° C. over the T_(max) of theanalogous unmodified complexes. An implication of this finding is thatreplacement of a phosphate oxygen atom with sulfur does not seem tochange the geometry of the minor groove of AT-rich regions, which isstill optimal for binding the CDPI₃ moiety.

The tethered CDPI₃ has almost no effect on RNA.RNA duplexes, which areknown to adopt the A-form in aqueous solutions and have a very broadminor groove. For example, addition of a CDPI₃ residue to the 3′-end ofeither strand of a 2′-O-Me-r(pT)₈.2′-O-Me-r(pA)₈ duplex showed a modestpositive effect on T_(max) of 4-9° C. The geometry of RNA.DNA hybrids issomewhere between the A- and B-duplex configurations and both of the2′-O-Me-RNA.DNA duplexes studied here showed a substantial level ofMGB-assisted stabilization, although with some backbone preference.Tethering the MGB residue to the 2′-O-Me-RNA strand was more beneficial,providing an increase in T_(max) of 18° C. for 2′-O-Me-r(pT)₈.d(pA)₈and >21-22° C. for the d(pT)₈.2′-O-Me-r(pA)₈ duplex. In contrast, alower effect on stabilization (ΔT_(max)=7° C.) was found when the CDPI₃residue was bound to the d(pA)₈ strand. CDPI₃-tailed d(pT)₈ was unusualin that conjugation of the MGB to the 5′-end of octadeoxythymidylateprovided >19° C. stabilization for its duplex with 2′-O-Me-r(pA)₈,whereas 3′-CDPI₃ had almost no effect on stability of this complex. Goodagreement of these data with our previously reported results obtained onpoly(rA).d(pT)₈ (Lukhtanov et al. (1995, 1996) supra) indicates thataddition of a methyl group on a 2′-OH in the minor groove of an RNA.DNAduplex does not substantially alter binding properties of the conjugatedCDPI₃ residue. Similar results have been obtained for unconjugatedCC-1065 bound to AT-rich sites in duplexes with varying backbonestructures. Kim et al. (1995) Antisense Res. Dev. 5:49-57.

TABLE 7 Melting temperatures (±1° C.) of duplexes formed byoctathymidylate and octa-adenylate with different backbone modificationsand CDPI₃ residues attached to different ends. Octa-adenylatederivatives: Octathymidylate derivatives 2′-O-Methyl PhosphorothioateType of 3′- and DNA RNA DNA backbone 5′-tails^(a) 3′-HEX 5′-Hex-NH₂3′-CDPI₃ 5′-CDPI₃ 5′,3′-di-CDPI₃ 3′-HEX-OH 3′-CDPI₃ 3′-Hex-NH₂ 3′-CDPI₃DNA 3′-Hex-NH₂ 14 12 58 58 55 12 30 <0 41 5′-Hex-NH₂ 13 13 58 56 44 1028 <0 42 3′-CDPI₃ 61 61 71 67 64 19 50 49 61 5′-CDPI₃ 53 53 63 74 61 1749 41 56 5′,3′-di- 60 57 69 68 71 — — — — CDPI₃ 2′-O-Methyl 3′-Hex-OH <0<0 ~0 19 — 20 29 ND^(b) ND^(b) RNA 3′-CDPI₃ 22 21 54 58 — 24 41 <0ND^(b) Phospho- 3′-Hex-NH₂ 8 9 55 55 — 34 48 <0 35 rothioate 3′-CDPI₃ 5556 70 73 — 43 65 40 57 DNA ^(a)The oligonucleotides with thismodification have a terminal phosphate linked to the hydroxy group of1,6-hexanediol (Hex-OH) or 6-amino-1-hexanol (Hex-NH₂) residues. Thestructure of the CDPI₃ residue and linkers for 3′- and5′-oligonucleotide conjugates are shown in FIG. 2. ^(b)No meltingtransition detected.

TABLE 8 Structure and stability of tetradecanucleotide duplexes modifiedby a CDPI₃ residue T_(max) Duplex structure 3′-Tail (° C. ± 1° C.)5′-d(GpTpGpTpGpTpCpApTpApTpApTpAp)-X-3′ X = —O(CH₂)₆NH₂ 48 (SEQ ID No.:31) NH₂(CH₂)₆O-d(pCpApCpApCpApGpTpApTpApTpApT)-5′ X = —O(CH₂)₆NH—CDPI₃69 (SEQ ID No.: 32) 5′-d(GpTpGpTpGpTpCpApTpApApApTpAp)-X-3′ X= —O(CH₂)₆NH₂ 49 (SEQ ID No.: 33) 3′-d(CpApCpApCpApGpTpApTpTpTpApT)-5′ X= —O(CH₂)₆NH—CDPI₃. 71 (SEQ ID NO: 34)

TABLE 9 Melting temperatures (±1° C.) of GC-rich octanucleotide duplexeswith CDPI₃ residues attached to the ends ApGpCpGpGpApTpG (strand B)CpApTpCpCpGpCpT 2′-O-Methyl RNA (strand A) DNA 5′-CPDI₃ PhosphorothioateDNA Type of 3′- and 3′-Hex- 3′-HEX- and Unmodi- backbone: 5′-tails^(a):NH₂ 5′-CDPI₃ 3′-CDPI₃ 5′,3′-di-CDPI₃ OH 3′-Hex-OH 3-CDPI₃ fied^(b)5′-CDPI₃ 3′-CDPI₃ DNA 3′-Hex-NH₂ 41 45 52 50 46 48 47 33 27 40 5′-CDPI₃58 76 79 76 52 71 80 49 70 75 3′-CDPI₃ 57 78 81 77 51 68 72 50 73 773′,5′-di- 60 BT^(d) 72 65 — — — — — — CDPI₃ 2′-O-Methyl 3′-Hex-OH 37 2029 — 66 67 67 28 ND^(c) ND^(c) RNA 5′-CDPI₃ 44 69 70 — 68 ~95 87 41 5864 and 3′-Hex-OH 3′-CDPI₃ 44 71 BT^(d) — 72 90 82 37 58 40 Phospho-Unmodi- 32 32 43 — 38 43 39 24 16 28 rothioate fied^(b) 5′-CDPI₃ 38 6769 — 38 64 66 28 62 63 DNA 3′-CDPI₃ 45 71 74 — 44 75 53 36 64 69^(a)Structures of the CDPI₃ residue and linkers for 3′- and5′-oligonucleotide conjugates are shown in FIG. 2. ^(b)These ODNs haveno tails. ^(c)No melting transition was detected. ^(d)Melting transitionwas too broad for T_(max) to be accurately determined.

Effect of Addition of a MGB Residue to a GC-Rich Octanucleotide Duplex

It is well recognized that A/T preference dominates the bindingspecificity of most MGBs, including CDPI oligomers. This preference islikely due to the hydrophobicity, depth and narrow width of the groove.Together these provide a perfect isohelical and van der Waals fit of thecrescent-shaped MGB molecules in the minor groove. Free CDPI₃ was shownto bind not only to poly(dA).poly(dT) but also to poly(dG).poly(dC),although with a lower strength. Boger et al. (1992) J. Org. Chem.57:1277-1284. Therefore, it was interesting to investigate the abilityof the CDPI₃ residue to stabilize short GC-rich and mixed duplexes.Table 9 shows the effect of the same MGB modifications discussed aboveon GC-rich octanucleotide duplexes, in which the nature of the minorgroove is altered. The test sequence, with a 3′-hexylamino tail, gave aduplex T_(max) of 41° C. Addition of a single MGB to either end ofstrand A increased the T_(max) by 16-17° C., while addition to strand Bgave a smaller increase in T_(max) (ΔT_(max)=4-11° C.). Strand B showeda preference for the position of CDPI₃ conjugation, with its3′-CDPI₃-tailed conjugate forming a more stable duplex (T_(max)=52° C.)than its corresponding 5′-CDPI₃ derivative (T_(max)=45° C.). This effectwas seen for all of the other duplexes presented in Table 9 (except whenstrand B has a 2′-O-methyl backbone) and could be due to the presence ofthe two AT pairs in the test sequence proximal to the site ofconjugation of the MGB.

Addition of the 2′-O-methyl modification (Table 9) to both strands gavea 25° C. increase in T_(max) over the 2′-deoxy strands. This haspreviously been shown to be a stabilizing modification. Inoue et al.(1987) Nucleic Acids Res. 15:6131-6148. A single MGB tethered to strandB did not change the T_(max) and addition to strand A gave a modestincrease (ΔT_(max)=2-6° C.). Hybrids between one strand bearing the2′-O-methyl modification and one with a DNA backbone were similar tothose of an unmodified DNA duplex (T_(max)=41° C.), with duplexstability lower when strand A was 2′-O-methyl (T_(max)=37° C.) andhigher when strand B was 2′-O-methyl (T_(max)=46° C.). Interestingly,CDPI₃ conjugation destabilized the former of these duplexes. This effectwas observed for both 3′-(T_(max)=29° C.) and 5′-CDPI₃-tailed(T_(max)=20° C.) derivatives of strand B (the DNA strand). Thisdestabilizing effect was exacerbated by phosphorothioate modification ofstrand B. In both cases, no detectable melting transition over 0° C. wasdetected, compared with a T_(max) of 28° C. for the non-conjugatedcounterpart.

Conversion of both backbones of the DNA octameric duplex to allphosphorothioate linkages reduced the T_(max) by 17° C. (from 41° C. to24° C.). Addition of a single MGB to strand B gave little change inT_(max) (even a decrease of 8° C. in the 5′-CDPI₃ case) and addition tostrand A gave a modest increase of 4-12° C. In general, phosphorothioateanalogs of strands A and B demonstrated hybridization properties similarto those observed for phosphodiester ODNs except that all of theircomplementary complexes have lower T_(max).

The conjugated CDPI₃ residue stabilized GC-rich DNA duplexes, with theextent of stabilization being about half, in terms of enhancement ofT_(max), of that observed for the d(pT)₈.d(pA)₈ complex. In contrast,another type of conjugated MGB, N-methylpyrrole carboxamide (MPC)oligomers, failed to stabilize the same GC-rich octadeoxyribonucleotideduplex used in this study. Sinyakov et al. (1995) J. Am. Chem. Soc.117:4995-4996. Without wishing to be bound by theory, CDPI₃ may be lesssensitive to the structure of the minor groove of a duplex than thenetropsin-type MPC peptides, because it does not form any hydrogen bondswith the bases and therefore the interaction is driven by van der Waalscontacts or hydrophobic forces. A narrower minor groove may promotebetter CDPI₃ binding and hence greater duplex stabilization.

CDPI₃-Conjugated Duplexes Containing Deoxyinosine in Place ofDeoxyguanosine

Substitution of deoxyguanosine (dG) by deoxyinosine (dI) in the modifiedODN could create a minor groove environment more suitable for CDPI₃binding, as was observed for netropsin and Hoechst 33258 (44). Nielsen(1991) Bioconjugate Chem. 2:1-12; Wartell et al. (1974) J. Biol. Chem.249:6719-6731; Marck et al. (1982) Nucleic Acids Res. 10:6147-6161; andMoon et al. (1996) Biopolymers 38:593-606. dI-containing analogues ofthe GC-rich duplex were prepared and studied with respect toMGB-assisted stabilization (Table 10). Replacement of the single dG ofstrand A with a dI residue gave a 10° C. decrease in T_(max). Additionof a single MGB to the 3′-end of either strand of this complex raisedthe T_(max) to a value 7-13° C. higher than the parent dG-containingduplex.

The effect of the tethered MGB on the duplex containing four dI residuesin strand B was dramatic. The duplex formed between this modified strandB and either the native or dI-substituted analog of strand A was weak(T_(max)=11° C. for native strand A) or nonexistent (for dI-substitutedstrand A). Addition of the MGB to the 3′-end of strand A raised theT_(max) from 11° C. to 48° C.; while addition of a MGB to the 3′-end ofstrand B raised the T_(max) from essentially 0° C. to 41° C. Thesevalues represent a 37-48° C. increase in T_(max) due to conjugation of aMGB to oligonucleotides forming duplexes containing dI.dC base pairs,which is close to the stability of the analogous dg-containing nativestrands. This shows that the conjugated CDPI₃ residue stabilizesdIdC-rich sequences as well as those rich in dAdT.

Duplexes with Two or More Conjugated CDPI₃ Residues

The ability of the minor groove of double-stranded DNA to bind two MGBresidues in a side-by-side antiparallel orientation has been noted.Kubista et al. (1987) Biochemistry 26:4545-4553; Mohan et al. (1992) J.Biomol. Struct. Dyn. 9:695-704; Fagan et al. (1992) J. Am. Chem. Soc.114:1080-1081; Mrksich et al. (1992) Proc. Natl. Acad. Sci. USA89:7586-7590; and Chen et al. (1994) J. Am. Chem. Soc. 116:6995-7005.Side-by-side binding of two MGB moieties in the duplex minor grooveaffords hyperstabilization in all duplexes studied which carry two CDPI₃residues tethered to opposite strands (Tables 7 and 9). For example, ad(pT)₈.d(pA)₈ duplex, already stabilized by 40-49° C. by one conjugatedCDPI₃ residue, was further stabilized by an additional 5-18° C. to reacha T_(max) of 74° C. for d(pT)₈.d(pA)₈ (Table 7). This is unprecedentedfor short duplexes with a phosphodiesler backbone. The greateststabilization occurred when either the 5′- or 3′-end of both strands wasmodified with an MGB; these could bind in the minor groove in anantiparallel mode (T_(max)=71 and 74° C.). The parallel orientation wasless beneficial (T_(max)=63 and 67° C.). This is consistent withliterature data for ‘free’ MGBs in which only the antiparallelorientation was experimentally observed. Mohan, supra; Fagan, supra;Mrksich, supra; and Animati et al. (1995) J. Med. Chem. 38:1140-1149.

This hyperstabilization did not seem to depend on either sequence orbackbone modification. All AT- and GC-rich duplexes that contained twoMGB residues, with one conjugated to each of the opposite strands, weresubstantially stabilized compared with analogous duplexes bearing asingle CDPI₃ tail (Tables 7 and 9). For example, Table 7 shows thatcomplexes formed by phosphorothioate analogs of d(pT)₈ and/or d(pA)₈possessing two CDPI₃ residues showed a T_(max) in the same range(T_(max)=56-73° C.) as their phosphodiester counterparts (T_(max)=63-74°C.). Addition of a MGB to both strands of a duplex with 2′-O-methylmodifications increased the stability by 21° C. (from 20° C. to 41° C.,Table 7). In the case of GC-rich duplexes, the stabilization resultingfrom conjugation of a second CDPI₃ residue to an opposite duplex strandwas even greater than that observed for the first CDPI₃ incorporation.For instance, attachment of one CDPI₃ residue increased stability of theGC DNA duplex by 4-17° C. and addition of the second CDPI₃ moietycontributed 18-33° C. to T_(max).

The data on duplexes with multiple conjugated MGBs in Tables 7 and 9show the following trends. If the duplex bore two CDPI₃ residuestethered to the same strand at the 3′- and 5′-ends almost no advantagein stability versus the corresponding mono-CDPI₃-tailed duplex was seen.This implies a strong hydrophobic interaction between two CDPI₃ residuesoccupying the same site in the minor groove of a short duplex.Furthermore, additional binding between the two MGB moieties attached tothe opposite duplex strands appears to add significantly to hybridstability. Similar effects of interaction of pendant hydrophobic groupson duplex and triplex stabilization were seen with ODNs conjugated tocholesterol residues. Gryaznov et al. (1993) Nucleic Acids Res.21:5909-5915. Addition of third and fourth CDPI₃ conjugated residuesnormally had no or a slightly negative effect on stability of theGC-rich duplexes studied (Table 9).

TABLE 10 Melting temperatures (±1° C.) of dG- and dI-containingoctanucleotide duplexes carrying a 3′-CDPI₃ residue Strand Bd(AGCGGATG)p d(AICIIATT)p 3′- 3′- 3′- Hex- 3′- Hex- 3′- Strand A:Tails^(a) NH₂ CDPI₃ NH₂ CDPI₃ d(CATCCGCT)p 3′-Hex- 41 52 11 — NH₂3′-CDPI₃ 57 81 48 67 d(CATCCICT)p 3′-Hex- 31 48 ~0 41 NH₂ 3′-CDPI₃ 54 7948 63 ^(a)Structures of the CDPI₃ residue and linker for the conjugatesare shown in FIG. 1.

Example 5 Reduced Dependence of T_(m) on Base Composition ofMGB-Oligonucleotide Conjugates

Short oligonucleotides of differing A+T content, conjugated to a MGB,were used to investigate the effect of base composition on T_(m) forMGB-short oligonucleotide conjugates (Table 11). T_(m)s were determinedon a Perkin Elmer λ2S UV/VIS spectrophotometer, equipped with a PTP-6temperature controller, using the PECSS software package. The A+Tcontent of the oligonucleotides tested ranged from 12.5% to 100% andT_(m) was determined for the hybrid of each of the oligonucleotides withits exact complement. As shown in Table 11, 8-mer MGB-oligonucleotideconjugates with A+T contents between 37.5% and 100% had T_(m)s thatranged between 45-54° C., a 9-degree span; while the T_(m)s forunconjugated 8-mers having similar A+T contents ranged over 38° C.

Other experiments have shown that, for 7-mers, T_(m)s ofoligonucleotides with A+T contents between 28% and 100% varied over arange of only 4.4° C. These results and those shown in Table 11 suggestthat, for short oligonucleotides conjugated to a MGB, T_(m) is moreclosely related to length than to base composition.

In addition, the range of T_(m) values for MGB-conjugated 8-mers extendsfrom 45° C., for oligonucleotides having a base composition of 100% A+T,to 63° C., for 0% A+T. Thus, the T_(m) values for MGB-conjugated 8-mersrange over approximately 18° C. By comparison, the T_(m) range forunconjugated 8-mers (between 0 and 100% A+T) encompasses at least 52° C.(Table 11). There is therefore a clear trend toward lessening of thedifferences in T_(m) between short oligonucleotides of different basecompositions, when such short oligonucleotides are conjugated to a MGB.

Example 6 Retention of Mismatch Discriminatory Capability of a ShortOligonucleotide Conjugated to a MGB

Although the dependence of T_(m) on base composition is suppressed forshort, 8-mer oligonucleotides that are conjugated to a MGB; theirheightened discriminatory ability, compared to unconjugatedoligonucleotides, is retained. Table 12 shows examples of T_(m)determinations for an 8-mer MGB-conjugated oligonucleotide hybridized toa perfectly-matched sequence and to four other sequences, eachcontaining a different single-nucleotide mismatch. The minimumdifference in T_(m) between a perfect match and a single-nucleotidemismatch is 19° C., while the maximum difference is 41° C.

TABLE 11 Effect of MGB conjugation on T_(m) values of 8-merMGB-oligonucleotide conjugates with varying A + T content T_(m) Sequence+MGB −MGB 5′-MGB-CAGCGGCG 63 52 5′-MGB-CAGCGACG 53 45 5′-MGB-CAGTGACG 4938 5′-MGB-CAGTGACA 47 33 5′-MGB-CAITIACA 53 17 5′-MGB-CAATGACA 54 275′-MGB-CAATGATA 45 20 5′-MGB-CAATAATA 45 12 5′-MGB-TAATAATA 45 <0

TABLE 12 Mismatch discrimination by 8-mer MGB-oligonucleotide conjugatesSequence T_(m) 5′-TTTTGTCACTGTTT (SEQ ID NO.:35) 47 ACAGTGAC-MGB-5′5′-TTTTGTCATTGTTT (SEQ ID NO.:36) 20 ACAGTGAC-MGB-5′ 5′-TTTTGTTACTGTTT(SEQ ID NO.:37) 25 ACAGTGAC-MGB-5′ 5′-TTTTGACACTGTTT (SEQ ID NO.:38) 28ACAGTGAC-MGB-5′ 5′-TTTTATCACTGTTT (SEQ ID NO.:39) 6 ACAGTGAC-MGB-5′

While the foregoing invention has been described in some detail by wayof illustration and example for purposes of clarity of understanding, itwill be apparent to those skilled in the art that various changes andmodifications may be practiced without departing from the spirit of theinvention. Therefore the foregoing descriptions and examples should notbe construed as limiting the scope of the invention.

1. A method for hybridizing nucleic acids, comprising the steps of: (a)providing a first nucleic acid and a second nucleic acid, (b) incubatingthe nucleic acids under hybridization conditions, and (c) identifyinghybridized nucleic acids; wherein at least one of the nucleic acidscomprises a minor groove binder (MGB)-oligonucleotide conjugate; whereinthe minor groove binder is a molecule having a molecular weight ofapproximately 150 to approximately 2,000 Daltons that binds in anon-intercalating manner into the minor groove of a double-strandednucleic acid with an association constant of greater than approximately10³M⁻¹.
 2. The method according to claim 1, wherein theMGB-oligonucleotide conjugate is a probe comprising a detectable label.3. The method according to claim 2, wherein the detectable label is afluorescent label.
 4. The method according to claim 3, wherein theMGB-oligonucleotide conjugate further comprises an agent that quenchesthe emission of the fluorescent label.
 5. The method according to claim4, further comprising the step of altering the spatial relationshipbetween the fluorescent label and the agent which quenches the emissionof the fluorescent label.
 6. The method according to claim 2, whereinthe method further comprises the step of releasing label from the probesubsequent to hybridization.
 7. The method according to claim 5, whereinthe method further comprises the step of releasing label from the probesubsequent to hybridization.
 8. The method according to claim 6, whereinrelease of label occurs as a result of exonuclease hydrolysis.
 9. Themethod according to claim 7, wherein release of label occurs as a resultof exonuclease hydrolysis.
 10. The method according to claim 3, whereinmore than one probe is used.
 11. The method according to claim 10,wherein a first and second probe is used.
 12. The method according toclaim 11, wherein the first probe comprises a fluorescence donor and thesecond probe comprises a fluorescence acceptor, and further wherein theemission wavelengths of the fluorescence donor overlap the absorptionwavelengths of the fluorescence acceptor.
 13. The method according toclaim 1 wherein the MGB-oligonucleotide conjugate is a primer comprisinga free 3′-hydroxyl group.
 14. The method according to claim 13, furthercomprising the step of extending the primer with a polymerizing enzyme.15. The method according to claim 14, wherein the polymerizing enzyme isa thermostable enzyme.
 16. The method according to claim 13, wherein theMGB-oligonucleotide conjugate is a primer in an amplification reaction.17. The method according to claim 16, wherein the amplification reactionis a polymerase chain reaction.
 18. A method for primer extension,comprising the steps of: (a) providing a sample containing a targetsequence, (b) providing one or more oligonucleotide primerscomplementary to regions of the target sequence, (c) providing apolymerizing enzyme and nucleotide substrates, and (d) incubating thesample, the oligonucleotide primers, the enzyme and the substrates underconditions favorable for polymerization; wherein at least one of theprimers comprises a MGB-oligonucleotide conjugate.
 19. The methodaccording to claim 18, wherein the method is an amplification reaction.20. The method according to claim 19, wherein the amplification reactionis a polymerase chain reaction.
 21. A method for discriminating betweenpolynucleotides which differ by a single nucleotide, the methodcomprising the following steps: (a) providing a polynucleotidecomprising a target sequence, (b) providing at least twoMGB-oligonucleotide conjugates, wherein one of the at least twoMGB-oligonucleotide conjugates has a sequence that is perfectlycomplementary to the target sequence and at least one other of theMGB-oligonucleotide conjugates has a single-nucleotide mismatch with thetarget sequence; (c) separately incubating each of theMGB-oligonucleotide conjugates with the polynucleotide underhybridization conditions; and (d) determining the hybridization strengthbetween each of the MGB-oligonucleotide conjugates and thepolynucleotide.
 22. A method for discriminating between polynucleotideswhich differ by a single nucleotide, the method comprising the followingsteps: (a) providing a MGB-oligonucleotide conjugate of definedsequence, (b) providing at least two polynucleotides, each of whichcomprises a target sequence, wherein one of the polynucleotides has atarget sequence that is perfectly complementary to theMGB-oligonucleotide conjugate and at least one other of thepolynucleotides has a target sequence having a single-nucleotidemismatch with the MGB-oligonucleotide conjugate; (c) separatelyincubating each of the polynucleotides with the MGB-oligonucleotideconjugate under hybridization conditions; and (d) determining thehybridization strength between each of the polynucleotides and theMGB-oligonucleotide conjugate.
 23. A method of ligating two or moreoligonucleotides, each of which is hybridized to adjacent sites on atarget nucleic acid, comprising the steps of: (a) providing a samplecontaining a target sequence, (b) providing at least twooligonucleotides which are complementary to adjacent sites on the targetsequence, (c) incubating the sample and the oligonucleotides underconditions favorable for ligation, and (d) identifying ligated nucleicacids; wherein at least one of the oligonucleotides comprises aMGB-oligonucleotide conjugate.
 24. An oligonucleotide probe comprising a5′-end, a 3′-end and one or more detectable labels, wherein the probe isa MGB-oligonucleotide conjugate.
 25. The probe according to claim 24wherein the detectable label is a fluorescent label.
 26. The probeaccording to claim 25 wherein the label is a fluorescein.
 27. The probeaccording to claim 25 wherein the label is a cyanine.
 28. The probeaccording to claim 25 wherein the label is a rhodamine.
 29. The probeaccording to claim 24 wherein the MGB is located at the oligonucleotide5′ end.
 30. The probe according to claim 24 wherein the MGB is locatedat the oligonucleotide 3′ end.
 31. The probe according to claim 24wherein the label is located at the oligonucleotide 5′ end.
 32. Theprobe according to claim 24 wherein the label is located at theoligonucleotide 3′ end.
 33. The probe according to claim 24 wherein theMGB is selected from the group consisting of a trimer of1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate (CDPI₃) and apentamer of N-methylpyrrole-4-carbox-2-amide (MPC₅).
 34. The probeaccording to claim 25 comprising multiple fluorescent labels.
 35. Theprobe according to claim 34 wherein the emission wavelengths of one ofthe fluorescent labels overlaps the absorption wavelengths of another ofthe fluorescent labels.
 36. The probe according to claim 25 furthercomprising a quenching agent which quenches the fluorescence emission ofthe fluorescent label.
 37. The probe according to claim 36 wherein thefluorescent label is a fluorescein.
 38. The probe according to claim 37wherein the quenching agent is tetramethylrhodamine.
 39. AMGB-oligonucleotide conjugate for use as a primer comprising a 5′ endand a 3′ end, wherein the 3′ end is extendible by a polymerizing enzyme.40. The primer according to claim 39, wherein the 3′ end comprises afree 3′ hydroxyl group.
 41. The primer according to claim 39, whereinthe primer has been extended by a polymerizing enzyme.
 42. The primeraccording to claim 41, wherein the primer has been extended during thesynthesis of a cDNA molecule.
 43. The primer according to claim 41,wherein the primer has been extended during an amplification reaction.44. The probe according to claim 24, wherein inosine is substituted forguanosine.
 45. The probe according to claim 24, wherein6-amino-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one is substituted forguanine.
 46. The probe according to claim 24, wherein4-amino-1H-pyrazolo[3,4-d]pyrimidine is substituted for adenine.
 47. Theprobe according to claim 24, wherein1H-pyrazolo[3,4-d]pyrimidin-4(5H)-6(7H)-dione is substituted foradenine.
 48. The primer according to claim 39, wherein inosine issubstituted for guanosine.
 49. The primer according to claim 39, wherein6-amino-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one is substituted forguanine.
 50. The primer according to claim 39, wherein4-amino-1H-pyrazolo[3,4-d]pyrimidine is substituted for adenine.
 51. Theprimer according to claim 39, wherein1H-pyrazolo[3,4-d]pyrimidin-4(5H)-6(7H)-dione is substituted foradenine.
 52. A composition comprising the probe according to claim 24.53. A composition comprising the primer according to claim
 39. 54. Theprobe according to claim 24, wherein the probe is less than 20nucleotides in length.
 55. The primer according to claim 39, wherein theprimer is less than 20 nucleotides in length.
 56. A kit foramplification comprising one or more primers according to claim
 39. 57.A kit for hybridization analysis comprising one or more probes accordingto claim
 24. 58. A kit for use in a hydrolyzable probe assay comprisingone or more probes according to claim
 24. 59. A kit for use in singlenucleotide mismatch detection comprising one or more probes according toclaim
 24. 60. A kit for use in single nucleotide mismatch detectioncomprising one or more primers according to claim
 39. 61. A kit for usein nucleotide sequence analysis comprising one or more of the probes ofclaim
 24. 62. A kit for use in nucleotide sequence analysis comprisingone or more of the primers of claim
 39. 63. A method forprimer-dependent nucleotide sequence analysis wherein aMGB-oligonucleotide conjugate is used as a primer.
 64. A method fordetermining the sequence of a polynucleotide comprising the steps of:(a) providing an array of oligonucleotide probes of different sequences,(b) incubating the polynucleotide and the array under hybridizationconditions, and (c) determining to which of the oligonucleotide probesin the array the polynucleotide hybridizes; wherein one or more of theoligonucleotide probes comprises a MGB-oligonucleotide conjugate.
 65. Amethod for examining gene expression comprising the steps of: (a)providing an array of oligonucleotide probes of different sequences, (b)incubating a population of polynucleotides with the array underhybridization conditions, and (c) determining to which of theoligonucleotide probes in the array the population hybridizes; whereinone or more of the oligonucleotide probes comprises aMGB-oligonucleotide conjugate.
 66. A method for identifying one or moremutations in a gene of interest comprising the steps of: (a) providingan array of oligonucleotide probes of different sequences, (b)incubating a polynucleotide sample with the array under hybridizationconditions, and (c) determining to which of the oligonucleotide probesin the array the polynucleotide hybridizes; wherein one or more of theoligonucleotide probes comprises a MGB-oligonucleotide conjugate. 67.The method of hybridization according to claim 1, wherein the meltingtemperature of the hybridized nucleic acid is independent of basecomposition.
 68. The method of hybridization according to claim 66wherein the melting temperature of the hybridized nucleic acid isdependent primarily on the length of the hybridized nucleic acid.
 69. AMGB-oligonucleotide conjugate that hybridizes to a target nucleic acidto form a hybrid, wherein the melting temperature of the hybrid isindependent of base composition.
 70. A method for cDNA synthesis,wherein a MGB-oligonucleotide conjugate is used as a primer.